DNA sequences involved in soraphen biosynthesis by myxobacteria

ABSTRACT

The present invention relates to a DNA molecule isolated from the genome of Sorangium cellulosum that encodes a polypeptide required for soraphen biosynthesis and to methods for the preparation of said DNA fragment. The present invention further relates to plasmids, vectors, and host cells that comprise the DNA molecule of the invention.

FIELD OF THE INVENTION

The present invention relates to the area of recombinant DNA technologyand in particular to the application thereof to myxobacteria, butpreferably to myxobacteria of the Sorangium/Polyangium group, for thelocalization, identification and cloning of individual genes and even ofentire gene clusters within the bacterial genome.

BACKGROUND OF THE INVENTION

The myxobacteria of the Sorangium/Polyangium group are highlyspecialized organisms which are frequently detectable in soil samples,dead plant material or in animal dung. Characteristic of this group ofmicroorganisms is, inter alia, their ability to utilize cellulose orcellulose-containing degradation products as sole C source. Anothercharacteristic feature of this group is their ability to produce highlyactive secondary metabolites.

To date, numerous strains of this group of organisms which are able, forexample, to synthesize plant-microbicidal compounds have been described.Of particular importance in this connection are the so-called soraphens,whose structural particulars are described in detail, for example, in EP0 358 606. The soraphens are macrocyclic compounds which have acytostatic activity and a favorable biocidal spectrum against pathogenicmicroorganisms, but especially against phytopathogenic fungi. Thesecompounds have very advantageous curative, systemic and, in particular,preventive properties and are therefore outstandingly suitable for usefor the therapeutic treatment of mammalians and protecting numerous cropplants.

It is also known of other representatives of the group of myxobacteriathat they are able to synthesize highly active compounds with antibioticand cytostatic potency Reichenbach et al (1988)!. Because of thepharmaceutical potential of these compounds and the importance,resulting therefrom, for plant protection, there is great interest inunderstanding the genetic bases of their synthesis in order thus toprovide the possibility of being able to influence them specificallywhere appropriate.

This is desirable in particular because the natural producer strains, asalso in the case of soraphen biosynthesis, very often provide theinteresting secondary metabolites only in inadequate concentrationswhich are far from sufficient to satisfy the demand for substance forwider-ranging activity assays, to say nothing of commercial production.

Furthermore, these strains prove in many cases to be problematic anddifficult to handle and therefore unsuitable for use on an industrialscale, for example in a fermentation process.

It would therefore be extremely desirable to decipher the genetic basesof secondary metabolite production in individual interestingrepresentatives of the myxobacteria, because this opens up a largenumber of possibilities for intervening directly or indirectly in thesynthetic process. Thus, for example, it would be conceivable to attemptto increase the productivity of the producer strains by targetedmutations or insertion of further gene copies. Another conceivablealternative relates to a possible transfer of the complete gene clusterwhich is responsible for secondary metabolite biosynthesis, or elseselected parts thereof, into an organism strain which is optimized forproduction purposes and which can then be employed for metaboliteproduction on the industrial scale.

However, the precondition for this is the provision of a method whichmakes it possible for these organisms to be subjected to direct andpreferably targeted genetic manipulation using recombinant DNAtechniques, for example by targeted incorporation of new genes or genefragments or other DNA sequences, including whole plasmids, into thegenome of the myxobacteria.

Individual representatives from the group of myxobacteria have alreadybeen the subject of investigations in this direction. Particularinterest was directed in this connection primarily at Myxococcusxanthus, a myxobacterium which has to date already been rather wellresearched and for which various gene transfer methods have also alreadybeen described. Thus, for example, the coli phage P1 was initiallyutilized very intensively for investigations relating to the insertionof transposon Tn5 into the Myxococcus xanthus chromosome Kaiser (1984);Kuner and Kaiser (1981)! and then subsequently also for transfer ofgenes cloned in Myxococcus xanthus back into the original E. coli hostO'Conner and Zusman (1983); Shimkets et al (1983)!.

Another method for gene transfer is based on the use of the plasmid RP4which has a very wide host range. Breton et al (1985) were able to showthat this plasmid can be transferred via conjugation from E. coli toMyxococcus xanthus and there undergoes stable integration into thechromosome. Based on these properties, Breton et al (1986) and Bretonand Guespin-Michel (1987) were able to integrate foreign genes into thechromosome of Myxococcus xanthus. As emerged from investigations byJaoua et al (1987; 1989), the observed integration is very probablybased on a so-called site specific recombination. The latter is thusconfined to particular sites, which have a narrow spatial limitation,within the Myxococcus xanthus chromosome because it is mediated by oneor more so-called hot spots on the RP4 plasmid. A transformation systemof this type is, because of the structural limitations, very limited inits flexibility and therefore unsuitable for use within the scope of thepresent invention.

Furthermore, it has emerged in the course of the investigations carriedout within the scope of this invention that the previously knownMyxococcus system discussed here is not applicable to bacteria of theSorangium/Polyangium group. It is assumed that these organisms lack thespecific structural elements which are necessary for site specificrecombination on their chromosomes. In addition, it has been found thatno stable transposition, for example on use of transposon Tn5, takesplace with these organisms either.

Pending EP-A No. 92810128.6 and Jaoua et al (1992) disclose for thefirst time a transformation system which makes possible direct andtargeted genetic manipulation of representatives of the group ofmyxobacteria, but especially of myxobacteria of the Sorangium/Polyangiumgroup, using recombinant DNA techniques and thus provides thepreconditions for the identification and isolation, disclosed within thescope of this invention, of DNA fragments which comprise at least onegene or parts of a gene which are involved directly or indirectly in thebiosynthesis of secondary metabolites, but especially in soraphenbiosynthesis.

The object which is to be achieved within the scope of this inventionthus primarily relates to the provision of a DNA fragment whichcomprises a DNA region which is involved directly or indirectly in thesynthesis of secondary metabolites in myxobacteria and in this casepreferably in myxobacteria of the Sorangium/Polyangium group, butespecially in the synthesis of soraphen, and which comprises at leastone of the involved genes or parts thereof, but preferably the totalityof the DNA sequences responsible for the secondary metabolite synthesis.

It has already been known for a long time from earlier investigations,for example on actinomycetes, that genes which are involved directly orindirectly in the individual steps of secondary metabolite biosynthesisare, in the majority of cases known hitherto, organized in the form ofgene clusters on the bacterial chromosome. By contrast, involvement ofplasmid-bound genes has been detectable to date only in rare cases.

A number of procedures have been proposed to date for isolating geneswhose gene products are involved indirectly or directly in antibioticbiosynthesis. These include, for example, the so-called complementationmethods in the course of which it is attempted to restore, with the aidof fragments obtained from wild-type DNA, the synthetic ability ofdefective mutants which, because of a specific mutation, are no longerable to synthesize the required antibiotic see, for example, Malpartidaand Hopwood, 1984!.

An alternative method for finding genes which are involved in thebiosynthesis of polyketide antibiotics is described in WO 87/03907. Thisentails using a DNA fragment which comprises at least one part of a genewhich is involved in the biosynthesis of a known polyketide antibioticas DNA hybridization probe for screening a genomic gene bank which haspreviously been prepared from the genomic DNA of the interestingmicroorganism.

Although the principal techniques for the identification and isolationof such gene clusters are thus known, the applicability thereof to anovel organism group which has been but little investigated to date is,as a rule, problematic because of the uncertainties involved. This isparticularly true when, as in the present case, there is no informationavailable about the structural organization of the genome and therefore,in the search for the gene cluster responsible for the secondarymetabolite biosynthesis, it is first necessary to find a suitablestarting point for the necessary genome analysis. This is probably alsothe reason why the relatively modest information concerning the geneticstructuring of secondary metabolite synthesis is to date concentrated ona few well-investigated organism groups, while next to nothing is knownfor others, such as, for example, the myxobacteria.

It has now been possible for the first time within the scope of thepresent invention, using the method described in EP-A No. 92810128.6 andJaoua et al (1992), to locate within a myxobacterial genome a DNA regionwhich is involved directly or indirectly in the biosynthesis ofsecondary metabolites and which is used as starting point for theidentification and isolation of the gene cluster surrounding this regionand subsequently to isolate and to clone the latter. The region is, inthis connection, in particular one which can be obtained from the genecluster, which is responsible for soraphen biosynthesis, within thegenome of S. cellulosum and which is demonstrably involved in thebiosynthesis of soraphen.

SUMMARY OF THE INVENTION

The present invention thus primarily relates to a DNA fragment whichcomprises a DNA region which is preferably obtainable from the genecluster, which is responsible for soraphen biosynthesis, within thegenome of S. cellulosum and which is involved directly or indirectly inthe biosynthesis of soraphen, including the adjacent DNA regions on theright and left, which, because of their function in connection withsoraphen biosynthesis, are revealed as constituents of the "soraphengene cluster" and which can be identified with the aid of the previouslyisolated DNA fragment.

Preferred within the scope of this invention is a DNA fragment whichcomprises a DNA region which is obtainable from the "soraphen genecluster" of S. cellulosum with the aid of the method describedhereinafter.

A particularly preferred DNA fragment comprises a 1.8 Kb DNA regionwhich is obtainable by the method according to the invention from theSorangium cellulosum genome and which comprises the nucleotidepart-sequence depicted in SEQ ID NO 1, as well as all other DNAsequences which are in the vicinity of this sequence and which, becauseof homologies which are present, can be regarded as structural orfunctional equivalents and therefore are able to hybridize with thissequence.

A DNA fragment which is likewise particularly preferred is one whichcomprises a 6.5 Kb DNA region which is obtainable by the methodaccording to the invention from the Sorangium cellulosum genome and hasthe restriction pattern depicted in FIG. 1 and additionally alsoincludes the 1.8 Kb region characterized in detail above.

A DNA fragment which exclusively comprises genomic DNA is veryparticularly preferred.

Likewise embraced by the present invention are DNA fragments whichcomprise sequence portions which have homologies with the nucleotidepart-sequence, depicted in SEQ ID NO 1, of the 1.8 Kb region which isobtainable by the method according to the invention from the Sorangiumcellulosum genome, which therefore can be found by use of this region orof parts thereof as hybridization probes within a genomic gene bank.

Preferred DNA fragments are likewise those which comprise sequenceportions which have homologies with the 4.6 Kb BamHI fragment from thegraI region of the granaticin gene cluster ORF 1-4! of Streptomycesviolaceoruber Tu22 and which therefore can be found by use of this 4.6Kb fragment or of parts of this fragment as hybridization probe within agenomic gene bank of a soraphen-producing organism.

Another aspect of the present invention relates to the use of a DNAfragment which comprises a portion of a gene cluster which isresponsible for soraphen biosynthesis, such as, for example, of one ofthe genes which is involved directly or indirectly in the biosynthesis,or parts thereof, as probe for finding adjacent overlapping DNA regionswhich can then in their turn again be used as probe for finding adjacentregions, which finally leads to complete identification of the genecluster which flanks the start or initial region and is responsible forsoraphen biosynthesis.

The present invention further relates to the DNA regions which can beobtained in the way described previously and which, if required, can beligated together to give a single fragment which then comprises allthose genes and other DNA sequences which flank within the Sorangiumgenome the firstly identified initial region in the form of a genecluster and which in their totality are responsible for soraphenbiosynthesis.

A preferred probe molecule is a DNA fragment which comprises a 1.8 KbDNA region which can be obtained by the process according to theinvention from the Sorangium cellulosum genome and which comprises thenucleotide part-sequence depicted in SEQ ID NO 1, but especiallyselected parts thereof. Particularly preferred sequence sections arethose which can be obtained from the flanking regions located on theright and left, respectively, of the said 1.8 Kb region.

Likewise preferred for use as a probe molecule is a 6.5 Kb fragmentwhich comprises a 6.5 Kb DNA region which can be obtained by the processaccording to the invention from the Sorangium cellulosum genome andwhich has the restriction pattern depicted in FIG. 1 and additionallyincludes the 1.8 Kb region characterized in detail above, but especiallyselected parts thereof. Particularly preferred sequence sections arethose which can be obtained from the flanking regions located on theright and left, respectively, of the 1.8 Kb region.

The present invention additionally relates to recombinant DNA moleculeswhich comprise one of the DNA fragments according to the invention, andto the plasmids and vectors derived therefrom. Likewise embraced arehost organisms which are transformed with said plasmid DNA or vectorDNA, including plant hosts.

The invention further relates to a method for the identification,isolation and cloning of a DNA fragment which comprises a DNA regionwhich can preferably be obtained from the gene cluster, which isresponsible for soraphen biosynthesis, within the genome of S.cellulosum and comprises at least one gene or a part of a gene oranother DNA sequence which is involved directly or indirectly in thebiosynthesis of soraphen, and which method essentially comprises thefollowing measures:

(a) construction of a representative gene library of asoraphen-producing organism from the group of myxobacteria, whichessentially comprises the totality of the bacterial genome distributedon single clones;

(b) screening of said clones using a specific DNA probe which hybridizesat least with a part of the gene cluster responsible for soraphenbiosynthesis;

(c) selection of those clones which exhibit a hybridization signal withthe DNA probe; and

(d) isolation of a DNA fragment from said clone, which comprises a DNAregion which comprises at least one gene or a pan of a gene or anotherDNA sequence which is involved directly or indirectly in thebiosynthesis of soraphen.

The present invention further relates to a method for the identificationand isolation of all those DNA sequences which are involved in theconstruction of the `soraphen gene cluster` flanking the initial orstart region, which comprises

(a) constructing a representative gene library of a soraphen-producingorganism from the group of myxobacteria, which essentially comprises thetotality of the bacterial genome distributed on single clones;

(b) hybridizing said clones using one of the previously isolated DNAfragments or selected parts thereof as probe molecules, which overlap atleast with a part of the adjacent DNA regions located on the right andleft, respectively, within the `soraphen gene cluster`;

(c) selecting those clones which exhibit a strong hybridization signalwith the DNA probe;

(d) isolating those fragments which comprise overlapping DNA regionsfrom the clones selected according to (c), and isolating the fragmentwhich projects furthest beyond the region of overlap;

(e) testing the DNA fragment isolated according to (d) for its abilityto function within the `soraphen gene cluster`;

(f) if it is possible to detect a function of the DNA fragment isolatedaccording to (d) within the scope of sorapben biosynthesis, repeatingthe method according to steps (a) to (e), wherein the DNA fragmentisolated according to (d), or selected parts thereof, but especiallythose from the left or right flanking region of said fragment, functionas DNA probes, until a function in soraphen biosynthesis is no longerdetectable within the scope of the function test on the particular newlyisolated DNA fragment, and thus the end of the gene cluster is reached;and

(g) carrying out the method according to steps (a) to (f) whereappropriate in the other direction which has not previously beenselected.

DETAILED DESCRIPTION OF THE INVENTION

In order to facilitate interpretation and understanding of what isintended to be regarded as encompassed according to the applicationwithin the scope of the present invention, some of the terms essentialfor establishing the scope of protection are defined in detailhereinafter, that is to say a detailed explanation is given of themeaning they are intended to have within the scope of this invention:

DNA fragment: A piece of DNA which may comprise both coding andnon-coding sections and which either can be obtained directly from anatural source or else can be prepared with the aid of recombinant orsynthetic techniques or else a combination of said techniques.

Coding DNA sequence: A DNA sequence which is composed of individualnucleotide constituents in accordance with the rules of the genetic codeand which comprises structural information and which, aftertranscription and translation have taken place, results in theproduction of a corresponding polypeptide.

Gene: A defined region which is located within a genome and which,besides the abovementioned coding DNA sequence, comprises other,primarily regulatory, DNA sequences which are responsible for thecontrol of the expression, that is to say the transcription andtranslation, of the coding portion.

Essentially homologous: This ten relates primarily to DNA and amino-acidsequences which must, because of the homologies present, be regarded asstructural and/or functional equivalents. The structural and/orfunctional differences between the relevant sequences, which should as arule be minimal, can have very different causes. Thus, for example,these may comprise mutations which occur naturally or else are inducedartificially, or else the differences to be observed compared with theinitial sequence are based on a specific modification which can beintroduced, for example, within the scope of a chemical synthesis.

Functional differences can be regarded as minimal when, for example, thenucleotide sequence coding for a polypeptide, or a protein sequence, hasessentially the same characteristic properties as the initial sequence,within the area of enzymatic activity, of immunological reactivity or,in the case of a nucleotide sequence, of gene regulation.

Structural differences can be regarded as minimal as long there is asignificant overlap or similarity between the different sequences, orthe latter have at least similar physical properties. The latterinclude, for example, the electrophoretic mobility, chromatographicsimilarities, sedimentation coefficients, spectrophotometric properties,etc. In the case of the nucleotide sequences, the agreement should be atleast 60%, but preferably 75% and, very particularly preferably, 90% andmore. In the case of the amino-acid sequence, the corresponding valuesare at least 70%, but preferably 80% and, particularly preferably, 90%.A 99% agreement is very particularly preferred.

Gene(s) or DNA of heterologous origin: A DNA sequence which codes for aspecific product or products or fulfills a biological function and whichoriginates from a different species than that into which the gene isinserted; said DNA sequence is also called foreign gene or foreign DNAor exogenous DNA.

Gene(s) or DNA of homologous origin: A DNA sequence which codes for aspecific product or products or fulfills a biological function and whichoriginates from the same species into which the gene is inserted. ThisDNA is also called exogenous DNA.

DNA homology: Degree of agreement between two or more DNA sequences.

Synthetic gene(s) or DNA: A DNA sequence which codes for a specificproduct or products or fulfills a biological function and which isprepared by a synthetic route.

Promoter: A DNA expression control sequence which ensures thetranscription of any desired homologous or heterologous DNA genesequence in a host cell as long as said gene sequence is linked in anoperable manner to a promoter of this type and the latter is active insaid host cell.

Termination sequence: DNA sequence at the end of a transcription unitwhich signals the end of the transcription process.

Overproducing promoter (OPP): Promoter which is able in a host cell tobring about the expression of any functional gene sequence(s) linked inan operable manner to an extent (measured in the form of the RNA or ofthe amount of polypeptide) which is distinctly higher than is naturallyobserved in host cells not transformed with said OPP.

3'/5' non-translated region: DNA sections which are locateddownstream/upstream of the coding region and which, although transcribedinto mRNA, are not translated into a polypeptide. This region comprisesregulatory sequences such as, for example, the ribosome binding site(5').

DNA expression vector: Cloning vehicle, such as, for example, a plasmidor a bacteriophage, which comprises all signal sequences which arenecessary for expression of an inserted DNA in a suitable host cell.

DNA transfer vector: Transfer vehicle, such as, for example, a plasmidor a bacteriophage vector, which makes it possible to insert geneticmaterial into a suitable host cell.

Homologous recombination: Reciprocal exchange of DNA fragments betweenhomologous DNA molecules.

Mutants, variants: Derivative, produced spontaneously or elseartificially using known process measures such as, for example, UVtreatment, treatment with mutagenic agents etc., of a microorganism,which still has the features and properties, essential to the invention,of the initial strain which this has received because of thetransformation with exogenous DNA.

To isolate the DNA fragments according to the invention, initiallygenomic gene banks are set up from the interesting organism strainswhich synthesize a required secondary metabolite, but especiallysoraphen.

Genomic DNA can be obtained in various ways from a host organism, forexample by extraction from the nuclear fraction and purification of theextracted DNA with the aid of known methods.

The fragmentation, which is necessary for setting up a representativegene bank, of the genomic DNA which is to be cloned to a size suitablefor inserting into a cloning vector can take place either by mechanicalshearing or else preferably by cutting with suitable restrictionenzymes. Particularly preferred in this connection within the scope ofthis invention is a partial cleavage of the genomic DNA, which leads tomutually overlapping DNA fragments.

Suitable cloning vectors which are already used routinely for producinggenomic and/or cDNA gene libraries comprise, for example, phage vectorssuch as the λ charon phages or else bacterial vectors such as the E.coli plasmid pBR322. Other suitable cloning vectors are known to theperson skilled in the art.

Particularly suitable for setting up genomic gene banks are so-calledcosmid vectors which comprise both portions from the DNA of plasmids andthe constituents, which are essential for packaging, from the genome oflambda phages, the so-called `cos` sites. The cosmids acquire, via theplasmid portion, gene sequences which code for one or more markers whichare required within the scope of selection, as well as the DNA regionswhich are essential for autonomous replication in a bacterial host.Cosmids are, as a rule, very small molecules, which provides them withthe capacity for cloning correspondingly large DNA fragments. This makesthem particularly suitable for use within the scope of the presentinvention.

Likewise suitable for setting up genomic gene banks are vector variantsderived from cosmids, such as, for example, the charomids, whichcomprise a variable amount of `full DNA` and therefore are able tocompensate for any differences in size with the DNA to be cloned.

Suitable clones which comprise the required genes or gene fragments canthen be discovered from the gene libraries set up in this way within thescope of a screening programme, for example with the aid of suitableprobe molecules DNA probes! and subsequently isolated. Various methodsare available for discovering suitable clones, such as, for example,differential colony hybridization or plaque hybridization. Whenexpression gene banks are used it is additionally possible to employimmunological detection methods which are based on identification of thespecific translation products.

It is possible to use as a probe molecule, for example, a DNA fragmentwhich has already been isolated previously from the same or else astructurally related gene which, because of the homologies present, isable to hybridize with the corresponding sequence section within therequired gene or gene cluster which is to be identified. Preferred foruse as probe molecule within the scope of the present invention is a DNAfragment which can be obtained from a gene or another DNA sequence whichplays a part in the synthesis of a known polyketide antibiotic. Aparticularly preferred probe molecule is a 4.6 Kb BamHI fragment fromthe graI region of the granaticin gene cluster ORF 1-4! of Streptomycesviolaceoruber Tu22 Sherman D. et al, 1989!.

In place of the probe from the graI region of Streptomyces violaceoruberTu22, which is used for finding a starting point within the `soraphengene cluster` and which exhibits only a relative weak hybridizationsignal, it is now also possible, of course, to use parts of thefragments isolated within the scope of the present invention, such as,for example, of the 1.8 Kb SalI fragment, as probe molecules, which,because of the homologies present, also leads to unambiguous resultswith other soraphen-producing Sorangium strains.

Once the amino-acid sequence of the gene to be isolated, or else atleast parts of this sequence, are known it is possible in an alternativeembodiment to design an appropriate corresponding DNA sequence on thebasis of this sequence information. Since, as is known, the genetic codeis degenerate, it is possible in the majority of cases to use differentcodons for one and the same amino acid. The result of this is that,apart from a few exceptional cases, as a rule a particular amino-acidsequence can be encoded by a whole series of mutually similaroligonucleotides. However, it should be noted in this connection thatonly one member of this series of oligonucleotides actually agrees withthe corresponding sequence within the gene which is sought. In order tolimit the number of possible oligonucleotides from the outset, it ispossible, for example, to have recourse to the rules established by BibbM. et al (1984) for the use of codons, which take account of thefrequency with which a particular codon is used in prokaryotic cellswith high guanine and cytosine content.

It is thus possible on the basis of this information to designoligonucleotide molecules which can be used as probe molecules foridentifying and isolating suitable clones, by hybridizing said probemolecules with genomic DNA in one of the previously described methods.

In order to facilitate the detectability of the required gene or elseparts of a required gene it is possible to label one of the previouslydescribed DNA probe molecules with a suitable, easily detectable group.A detectable group is intended to mean within the scope of thisinvention every material which has a particular, easily identifiable,physical or chemical property.

Materials of this type are already widely used, especially in the areaof immunoassays, and in the majority of cases can also be used in thepresent application. Particular mention may be made at this point ofenzymatically active groups such as, for example, enzymes, enzymesubstrates, coenzymes and enzyme inhibitors, furthermore fluorescent andluminescent agents, chromophores and radioisotopes such as, for example,³ H, ³⁵ S, ³² P, ¹²⁵ I and ¹⁴ C. The easy detectability of these markersis based, on the one hand, on their intrinsically present physicalproperties (for example fluorescence markers, chromophores,radioisotopes) and, on the other hand, on their reaction and bindingproperties (for example enzymes, substrates, coenzymes, inhibitors).

General methods relating to DNA hybridization are described, forexample, by Maniatis T et al (1982) and by Haymes B. T. et al (1985).

Those clones within the previously described gene libraries which areable to hybridize with a probe molecule and which can be identified withthe aid of one of the abovementioned detection methods can then befurther analyzed in order to determine the extent and nature of thecoding sequence in detail.

An alternative method for identifying cloned genes is based on theconstruction of a gene library which is constructed from expressionvectors. This entails, in analogy to the methods already describedpreviously, genomic DNA which is able to express a required gene productbeing initially isolated and subsequently cloned into a suitableexpression vector. The gene libraries produced in this way can then bescreened with the aid of suitable measures such as, for example, usingcomplementation studies, and those clones which comprise the requiredgene or else at least a part of this gene as insert can be selected.

It is thus possible, with the aid of the previously described methods,to isolate a gene which codes for a particular gene product.

For further characterization, the DNA sequences purified and isolated inthe manner previously described are subjected to a restriction analysisand to a sequence analysis.

The isolated fragments are characterized within the scope of therestriction analysis on the basis of their restriction cleavage sites,by being completed digested with suitable restriction enzymes such as,for example, BglII, SphI and SmaI. Said restriction enzymes can in thisconnection be employed either singly or else in combination with oneanother. The size of the resulting fragments can be determined afterfractionation on an agarose gel by comparison with a size standard.

For the sequence analysis, the previously isolated DNA fragments areinitially decomposed with the aid of suitable restriction enzymes intofragments and subsequently cloned into suitable cloning vectors such as,for example, the M13 vectors mp18 and mp19. The sequencing is carriedout, as a rule, in the 5'→3' direction, preferably using thedideoxynucleotide chain-termination method of Sanger Sanger et al, 1977!or the Maxam and Gilbert method Maxam and Gilbert, 1980!. In order toavoid errors in the sequencing, it is advantageous to sequence the twoDNA strands in parallel. Standardized sequencing kits which help toreduce the experimental effort and whose use is therefore preferredwithin the scope of this invention are now obtainable. The analysis ofthe nucleotide sequence and of the corresponding amino-acid sequence ispreferably carried out with computer assistance using suitable,commercially obtainable computer software for example GCG software fromthe University of Wisconsin!.

Various alternatives are available for analysis of the cloned DNAfragment with respect to its function within the scope of soraphenbiosynthesis.

Thus, for example, there is the possibility within the scope ofcomplementation experiments with defective mutants not only ofestablishing the involvement in principle of a gene or gene fragment insecondary metabolite biosynthesis but, in addition, of verifyingspecifically the synthetic step in which said DNA fragment is involved.

In an alternative form of analysis, the demonstration takes place inexactly the opposite sense. Transfer of plasmids which comprise DNAsections which have homologies with corresponding sections of themyxobacterial genome results in integration of said homologous DNAsections into the chromosomal DNA of the myxobacteria at the site of thehomology via homologous recombination. If the homologous DNA section is,as in the present case, a region within an intact gene cluster, theplasmid integration results in inactivation of this cluster by so-calledgene disruption and consequently in a suspension in secondary metaboliteproduction.

The bases for carrying out the detection method described above aredescribed in EP-A No. 92810128.6 and Jaoua et al (1992). It was possibleto show there for the first time that it is possible for foreign DNA ofhomologous or heterologous origin or a combination of genetic materialof homologous and heterologous origin to be inserted into themyxobacterial cell and there integrated, via homologous recombination,specifically at a site, which is accurately defined on the basis of thehomologies present, into the chromosome of said myxobacteria as long asthe latter have sufficient homologies with corresponding sections withinthe genome.

According to the present state of knowledge, it is assumed that ahomologous region which comprises at least 100 Bp, but preferably morethan 1000 Bp, is sufficient to bring about the required recombinationevent.

However, a homologous region which extends over a range from 0.3 to 4Kb, but in particular over a range from 1 to 3 Kb, is preferred.

Preferably provided for the preparation of suitable plasmids which havea homology with the myxobacterial chromosome which is sufficient forintegration via homologous recombination is a subcloning step in whichthe previously isolated cosmid DNA is digested and fragments of suitablesize are isolated and subsequently cloned into a suitable plasmid.

The ligation of homologous DNA fragments and of DNA fragments ofhomologous and heterologous origin into a suitable cloning vector takesplace with the aid of standard methods as are described, for example, byManiatis et al, 1982.

As a rule, this entails the vector and the DNA to be integratedinitially being cut with suitable restriction enzymes. Examples ofsuitable restriction enzymes are those which provide fragments withblunt ends, such as, for example, SmaI, HpaI and EcoRV, or else enzymeswhich form cohesive ends, such as, for example, EcoRI, SacI, BamHI,SalI, PvuI, etc.

Both fragments with blunt ends and those with cohesive ends which arecomplementary to one another can be linked again, with the aid ofsuitable DNA ligases, to give a single continuous DNA molecule.

Blunt ends can also be produced by treatment of DNA fragments which haveprotruding cohesive ends with the Klenow fragment of E. coli DNApolymerase by filling in the gaps with the appropriate complementarynucleotides.

On the other hand, cohesive ends can also be produced artificially, forexample by attaching complementary homopolymer tails to the ends of arequired DNA sequence and of the cut vector molecule using a terminaldeoxynucleotidyl-transferase or else by attaching syntheticoligonucleotide sequences (linkers) which have a restriction cleavagesite and subsequently cutting with the appropriate enzyme.

It is possible in principle to use for the preparation and replicationof the previously described constructs all conventional cloning vectorssuch as, for example, plasmid vectors or bacteriophage vectors as longas they have replication and control sequences which originate fromspecies which are compatible with the host cell.

As a rule, the cloning vector has an origin of replication, in additionspecific genes which lead to phenotypical selection features in thetransformed host cell, in particular to resistances to antibiotics. Thetransformed vectors can be selected on the basis of these phenotypicalmarkers after transformation into a host cell.

Selectable phenotypical markers which can be used within the scope ofthis invention comprise, for example, without this representing alimitation on the subject-matter of the invention, resistances toampicillin, tetracycline, chloramphenicol, hygromycin, G418, kanamycin,neomycin and bleomycin. A prototrophy for particular amino acids can,for example, function as further selectable marker.

Primarily preferred within the scope of the present invention are E.coli plasmids such as, for example, the plasmid pSUP2021 used within thescope of the present invention.

Primarily suitable as host cells for the previously described cloningwithin the scope of this invention are prokaryotes, including bacterialhosts such as, for example, A. tumefaciens, E. coli, S. typhimurium andSerratia marcescens, furthermore pseudomonads, actinomycetes,salmonellae and myxobacteria themselves.

E. coli hosts are particularly preferred, such as, for example the E.coli HB101 strain.

Competent cells of the E. coli HB101 strain are moreover produced withthe aid of the methods customarily used for transformation of E. colisee: "General recombinant DNA techniques"!.

The colonies resulting after transformation and subsequent incubation ona suitable medium are subjected to a differential screening by platingout on selective media. It is then possible subsequently to isolate theappropriate plasmid DNA from those colonies which comprise plasmids withDNA fragments cloned in.

Recombinant plasmids of various sizes are obtained in this way. It isthen possible after restriction analysis for plasmids of suitable sizeto be selected for the subsequent insertion of the plasmid DNA into themyxobacterial cell. This DNA transfer can moreover take place eitherdirectly or else via an intermediate host (donor cell) within the scopeof a conjugal transfer.

Conjugal transfer from a donor cell to the myxobacterial recipient ispreferred within the scope of this invention.

The DNA to be transferred within the scope of this conjugal transfer canmoreover be either initially cloned, as previously described, in one ofthe customarily used cloning vectors and subsequently transformed into asuitable intermediate host which functions as donor cell. However, thecircuitous route via the intermediate host can be avoided by using ahost strain which is suitable both for cloning of DNA and for use asdonor cell within the scope of the conjugation.

Intermediate hosts which can be used as donor cells within the scope ofthis invention are essentially prokaryotic cells selected from the groupconsisting of E. coli, pseudomonads, actinomycetes, salmonellae andmyxobacteria themselves.

The precondition for conjugal transfer of plasmid DNA from a donor cellto a recipient is the presence of transfer (tra) and mobilizationfunctions (mob). In this connection, the mobilization function mustcomprise at least the origin of transfer (oriT) and be located on theplasmid to be transferred. By contrast, the transfer function (tra) canbe located either on the plasmid or on a helper plasmid or else bepresent integrated into the chromosome of the donor cell.

Plasmids which meet the abovementioned precondition and are thereforepreferred within the scope of this invention essentially belong toincompatibility groups P, Q, T, N, W and ColI. The prototype of the Pgroup plasmids is the plasmid RP4. Particularly preferred within thescope of this invention is the plasmid pSUP2021 which comprises a 1.9 Kbfragment from the plasmid RP4 which has as constituent of the mobfunction (RP4mob) the origin of transfer (oriT). Other plasmids with themob function (RP4mob) such as, for example, pSUP101, pSUP301, pSUP401,pSUP201, pSUP202, pSUP203 or pSUP205, and the derivatives derivedtherefrom Simon et al (1988)!, can likewise be used within the scope ofthe method according to the invention.

It has proven advantageous to expose the myxobacterial recipient duringthe course of the conjugal transfer to a brief heat treatment beforeincubation with the donor strain. The recipient cells are preferablypreincubated at a temperature of 35° C. to 60° C., preferably at atemperature of 42° C. to 55° C. and very particularly preferably at atemperature of 48° C. to 52° C., for one to 120 minutes, but especiallyfor 5 to 20 minutes.

Used in a preferred embodiment of the present invention is a E. colidonor strain which comprises the transfer genes (tra) of plasmid RP4incorporated in the chromosomal DNA. The E. coli donor strain W3101(pME305) which comprises the helper plasmid pME305 which possesses thetransfer function (tra) of RP4 is particularly preferred within thescope of this invention.

Of particular interest for methodological techniques and therefore veryparticularly preferred within the scope of this invention are bacterialstrains which are suitable both as hosts for the cloning of vectors withintegrated DNA sequences and for use as donor cell within the scope ofthe conjugal transfer. Likewise particularly preferred are bacterialstrains which are restriction negative and thus do not degrade insertedforeign DNA. Both of the abovementioned criteria are met in an idealmanner by the E. coli strain ED8767 (pUZ8) which is, however, mentionedat this point only as representative of other suitable bacterial strainsand is not intended to limit the application in any way.

Besides the previously described conjugal gene transfer from a donorcell into a myxobacterial recipient, it is also, of course, possible touse other suitable gene transfer methods for inserting genetic materialinto myxobacteria. Mention may be made here primarily of gene transfervia electroporation, within the scope of which the myxobacterial cellsare briefly exposed to high electric field strengths Kuspa and Kaiser(1989)!. The general outline conditions for electroporation ofprokaryotic cells are described in detail in U.S. Pat. No. 4,910,140.

The DNA fragment according to the invention, which comprises a DNAregion which is involved directly or indirectly in the biosynthesis ofsoraphen and which can be obtained in the previously described way fromthe gene cluster of soraphen biosynthesis can also be used as startingclone for the identification and isolation of other DNA regions whichare adjacent and overlap with the latter from said gene cluster.

This can be achieved, for example, by carrying out a so-calledchromosome walking, using the previously isolated DNA fragment or elsein particular its 5'-and 3'-located flanking sequences, within a genelibrary composed of DNA fragments with mutually overlapping DNA regions.The procedure within the scope of chromosome walking are known to theperson skilled in this art. Details can be obtained, for example, fromthe publications by Smith C. L. et al (1987) and Wahl G. L. et al(1987).

The precondition for chromosome walking is the presence of clones withcoherent and mutually overlapping DNA fragments of maximum length withina gene library, and of a suitable starting clone which comprises afragment which is located in the vicinity of or else preferably withinthe region to be analyzed. If the exact location of the starting cloneis unknown, the walking is preferably carried out in both directions aswell.

The actual walking step starts by using the starting clone, once it hasbeen identified and isolated, as a probe in one of the previouslydescribed hybridization reactions in order to detect adjacent cloneswhich have regions overlapping with the starting clone. It is moreoverpossible by hybridization analysis to identify the fragment whichprojects furthest beyond the overlapping region. This fragment is thenused as the initial clone for the second walking step, and in this casethe fragment which overlaps with said second clone in the same directionis identified. In this way, by continuous advance along the chromosome,a collection of overlapping DNA clones which cover a large DNA range isobtained. These can then, where appropriate after one or else severalsubcloning steps have been carried out, be ligated together with the aidof known methods to give a fragment which comprises parts or else,preferably, all constituents essential for soraphen biosynthesis.

In place of the very large and unwieldy complete fragment, preferablyused for the hybridization reaction to identify clones with overlappingflanking regions is a partial fragment which is from the left or rightflanking region and which can be obtained by a subcloning step. Becauseof the smaller size of said partial fragment, the hybridization reactionresults in fewer positive hybridization signals so that the analyticaleffort is distinctly less than when the complete fragment is used. It isadditionally advisable to characterize the partial fragment in detail inorder to exclude the presence of relatively large portions of repetitivesequences, which may be scattered over the entire genome and thus wouldgreatly impede a targeted sequence of walking steps.

Since the gene cluster responsible for soraphen biosynthesis covers avery large genome region, it is advantageous within the scope of thepresent invention to carry out a so-called large-step walking or cosmidwalking. It is possible in these cases, by using cosmid vectors whichpermit the cloning of very large DNA fragments, to cover a very largeDNA region, which can comprise up to 45 Kb, with a single walking step.

In a specific embodiment of the present invention, to construct a cosmidgene bank of S. cellulosum complete DNA in a size of the order of 100 Kbis isolated and subsequently digested with the aid of suitablerestriction endonucleases.

Preferred in this connection is a 3-fold partial digestion with Sau3A,with the individual digestions being carried out independently of oneanother in order thus to achieve a restriction level with maximumdifferences.

The digested DNA is subsequently extracted in the customary way, inorder to remove the endonuclease which is still present, and isprecipitated and finally concentrated. The resulting fragmentconcentrate is then fractionated, for example via a density gradientcentrifugation, according to the size of the individual fragments.Fractions obtainable in this way can, after dialysis, be analyzed on anagarose gel. The fractions which comprise fragments of suitable size arepooled and concentrated for further processing. Regarded as particularlysuitable within the scope of its invention are fragments with a size ofthe order of 30 Kb to 45 Kb, but preferably from 40 Kb to 45 Kb.

In parallel with the fragmentation described above, or later, a suitablecosmid vector such as, for example, pHC79 Hohn and Collins, 1980! iscompletely digested with a suitable restriction enzyme such as, forexample, BamHI for the subsequent ligase reaction.

The ligation of the cosmid DNA to the S. cellulosum fragments which havebeen fractionated according to their size can be carried out using a T₄DNA ligase. The ligation mixture obtainable in this way is, after asufficient incubation time, packaged into λ phages, using, for reasonsof economy of the method, preferably one of the commercial packagingkits which can now be obtained from various suppliers such as, forexample, from STRATAGEN or PROMEGA.

The resulting phage particles are then used to infect a suitable hoststrain. A recA⁻ E. coli strain such as, for example, E. coli HB 101 ispreferred. The selection of transfected clones and the isolation of theplasmid DNA can be carried out by means of generally known methods.

The screening of the gene bank for DNA fragments which play a part insoraphen biosynthesis is carried out with the aid of a specifichybridization probe which is assumed to comprise DNA regions which havesufficient homologies with corresponding regions within the `soraphengene cluster`.

The starting material which can be used for preparing said hybridizationprobe is the plasmid pIJ5200 Sherman et al, 1989! which comprises a 4.6Kb BamHI fragment of the graI region granaticin gene cluster ORF 1-4! ofStreptomyces violaceoruber Tu22 cloned into the BamHI cleavage site ofpUC 18.

In order to remove interfering DNA sequences which originate from theplasmid vectors and which might have adverse effects on the gene bankanalysis from the 4.6 Kb, said fragment is preferably cloned into aStreptomyces vector and the latter is transformed into a competentStreptomyces strain.

After selection and restriction analysis, suitable clones which comprisethe 4.6 Kb fragment cloned into the plasmid DNA are selected andcultivated for plasmid isolation. The plasmid DNA is isolated with theaid of known methods such as, for example, the CsCl gradient method, andthe 4.6 Kb fragment is removed after complete digestion of the plasmidDNA with BamHI.

The 4.6 Kb fragment isolated in this way is radioactively labelled bycarrying out a nick translation using d-CTP³². It is advisable in thiscase too, for reasons of economy of the method, to use a commercial nicktranslation kit which is marketed, for example, by Bethesda ResearchLaboratories Life Technologies Inc.

It is now, of course, also possible to use as a probe molecule in placeof the probe which was used for finding a starting point within the`soraphen gene cluster` and which was obtained from the graI region ofStreptomyces violaceoruber Tu22, and which reveals only a relativelyweak hybridization signal, parts of the fragments isolated within thescope of the present invention, such as, for example, a 1.8 Kb SalIfragment, which leads, because of the homologies present, to unambiguousresults with other soraphen-producing Sorangium strains too.

The screening of the gene bank with the aid of the radioactivelylabelled probe preferably takes place within the scope of ahybridization analysis. The latter can be carried out, for example, inthe form of a colony hybridization which is described, for example, byManiatis et al page 326-328; (1982)!.

The clones which show the strongest hybridization signals are selectedand incubated on a suitable medium for example LB medium! and then usedto isolate the plasmid DNA as described by Maniatis et al (1982); pages368 and 369!.

The isolated plasmids are then digested with a suitable restrictionenzyme such as, for example, SalI, and the fragments obtained in thisway are fractionated on an agarose gel. The resulting fragments are thensubjected to a Southern hybridization which can be carried out withinthe scope of a Southern capillary blotting. A cosmid clone p98/1! whichshows a strong band at 1.8 Kb in the audioradiograph is selected forsubsequent work. This cosmid clone is first cultivated in a suitablemedium for amplification of the plasmid DNA. After the plasmid DNA hasbeen isolated it is completely digested with a suitable restrictionenzyme for example SalI! and the fragments obtainable in this way arefractionated by electrophoresis on an agarose gel. The required 1.8 KbDNA fragment can then be removed from the agarose gel by electroelution.

The 1.8 Kb fragment obtainable in this way is subsequently cloned into abacterial plasmid in a ligase reaction. The E. coli plasmid pBR322 ispreferred within the scope of this invention.

The resulting ligated DNA is subsequently cloned into cells, which havebeen made competent, of a recA⁻ strain of E. coli , but preferably intocells of the E. coli strain HB101 Maniatis et al, 1982; pages 250 and 251 ) and transferred to a selective medium.

Suitable colonies can be found by differential screening of theresulting transformed colonies, and their plasmid DNA can be isolated asdescribed by Maniatis et al, 1982; pages 368 and 369!. The isolatedplasmid DNA is then cut with a suitable restriction enzyme such as, forexample, SalI, and analyzed by agarose gel electrophoresis for the sizeof its inserted fragments, preferably employing as a comparison standardthe previously selected cosmid p98/1!.

A plasmid p108/III2! which comprises an additional fragment of therequired size can then be isolated from the gel in the manner previouslydescribed. The identity of this additional fragment to the 1.8 Kbfragment of the previously selected cosmid p98/1! can then be confirmedby Southern transfer and hybridization with the 4.6 Kb DNA probe from S.violaceoruber.

The function analysis of the previously isolated 1.8 Kb DNA fragment canbe carried out within the scope of a gene disruption experiment.

This entails initially the cosmid DNA p98/1! being completely digestedwith the aid of a suitable restriction enzyme, and the resultingfragments being fractionated, e.g. when using PvuI, fragments with sizesof about 10, 6.5, 4.2 and 4 Kb can be obtained. Fragments of therequired size can then be cloned into a suitable vector which comprisesthe functions required for conjugal transfer. The plasmid pSUP2021 whichcomprises a fragment from the plasmid RP4, which has a mob function(RP4mob) and as constituent of this mob function an origin of transfer(oriT), is preferred. After transformation of host cells which have beenmade competent,--which are preferably cells of the E. coli strainHB101--, it is then possible very simply to identify and select positiveclones, that is to say clones whose plasmid DNA comprises fragments ofthe required size.

The plasmids obtainable in this way can then be used for conjugaltransfer into S. cellulosum. The donor strain which can preferably beused for this is an E. coli strain which comprises the transfer genes(tra) of plasmid RP4 incorporated into the chromosomal DNA. Preferredwithin the scope of this invention is the E. coli donor strainW3101(pME305) which comprises the helper plasmid pME305 which possessesthe transfer function (tra) of RP4.

Of particular interest for methodological techniques and therefore veryparticularly preferred within the scope of this invention are bacterialstrains which are suitable both as hosts for the cloning of vectors withintegrated DNA sequences and for use as donor cells within the scope ofthe conjugal transfer. Likewise particularly preferred are bacterialstrains which are restriction negative and thus do not degrade insertedforeign DNA. Both of the previously mentioned criteria are fulfilled inan ideal manner by the E. coli strain ED8767(pUZ8) whose use istherefore particularly preferred within the scope of this invention.

When the helper plasmid pUZ8, which has no ampicillin-resistance gene,is used it is possible to dispense with the cloning step in the E. coliintermediate host HB101 because direct cloning in the E. coli donorstrain ED8767 which is intended for the conjugal transfer is nowpossible.

The plasmid pUZ8 is a derivative of the plasmid RP4 which embraces awide host range and is described by Datta et al (1971). Themodifications compared with the initial plasmid RP4 essentially relateto the ampicillin-resistance gene and to the insertion element IS21,both of which are deleted, and to the incorporation of an additionalgene which confers resistance to mercury ions see Jaoua et al (1987)!.

The plasmid DNA can therefore now be directly transformed into the E.coli strain ED8767. For this, competent cells of the E. coli strainED8767 are prepared with the aid of the methods customarily used for thetransformation of E. coli see: "General recombinant DNA techniques"!.

The colonies resulting after transformation and subsequent incubation ona suitable selective medium are subjected to a differential screening byparallel plating out on ampicillin-containing 60 μg/ml! andampicillin-free medium. It is subsequently possible to isolate thosecolonies which have lost their ampicillin resistance owing to theintegration of the Sorangium DNA fragments. The cultures obtainable inthis way can then be employed directly as donor cells for theconjugative transfer of the recombinant plasmids into Sorangiumcellulosum cells.

For the actual transfer, Sorangium cellulosum cells in the stationaryphase are mixed with a late log phase culture of E. coli donor cellswhich comprise a comparable proportion of cells.

It proves advantageous in this case to expose the Sorangium recipientcells to a brief heat treatment in a water bath before the conjugationwith E. coli . The best transfer results with the Sorangium cellulosumstrain SJ3 can be achieved with a heat treatment at a temperature of 50°C. for 10 minutes. Transfer frequencies of 1-5×10⁻⁵ can be achievedunder these conditions, which corresponds to an increase by a factor of10 compared with a method without previous heat treatment.

After various selection steps have been carried out it is possible toobtain transconjugants of Sorangium cellulosum which comprise therequired plasmid DNA. The transformation frequency for the transfer ofplasmid DNA to Sorangium averages 1-3×10⁻⁶ based on the recipientstrain.

Transfer of plasmids which comprise DNA sections which are homologouswith corresponding sections within the Sorangium cellulosum genome leadsto integration of said DNA sections into the chromosomal Sorangiumcellulosum DNA at the site of the homology via homologous recombination.If the homologous region is a section within a gene cluster, for examplethat for soraphen biosynthesis, the plasmid integration results ininactivation of this cluster by so-called gene disruption. This methodthus permits analysis of the function of a cloned DNA fragment inSorangium cellulosum.

To examine the function of the PvuI fragments of cosmid p98/1,well-grown transconjugant colonies are transferred to and cultivated ona selective medium which contains kanamycin phleomycin and streptomycinas selective agents.

One or more intermediate cultivation steps are followed by transfer to amedium which contains an adsorbent such as, for example, a polymericadsorber resin which binds the soraphen released by the bacteria.

After incubation for several days, the adsorbent is removed and theadherent soraphen is extracted, for example by shaking with a suitablesolvent such as, for example, isopropanol. Quantitative soraphenanalysis can then be carried out with the aid of an HPLC method,preferably using a UV detector at a wavelength of 210 nm.

For further characterization of the previously described 6.5 Kb fragmentthe latter is subjected to a restriction analysis, and subsequently the1.8 Kb fragment comprised in said 6.5 Kb fragment is sequenced.

To characterize the 6.5 Kb PvuI fragment on the basis of restrictioncleavage sites, said fragment is completely digested with suitablerestriction enzymes such as, for example, BglII, SphI and SmaI, whichare employed singly or in combination with one another. The size of theresulting fragments can be determined after fractionation on an agarosegel by comparison with a size standard.

Digestion of the 6.5 Kb PuvI fragment with BglII yields two fragments4.3 Kb and 2.3 Kb in size, and that with SphI yields 4 fragments 2.8 Kb,1.5 Kb, 0.7 Kb in size. Combination of the two enzymes BglII and SphI!results in 5 fragments 1.6 Kb, 1.5 Kb, 1.5 Kb, 1.2 Kb, 0.7 Kb in size.Digestion with SmaI provides three fragments which have a size of 2.9Kb, 2.0 Kb and 1.6 Kb.

In this case, because of the measurement method used, the actual size ofthe fragments may differ by ±10% from the stated value. The position ofthe BglII, SphI and SmaI cleavage sites on the 6.5 Kb fragment of cosmidp98/1 can be established on the basis of the fragment sizes found, asdepicted in FIG. 1.

To prepare for the subsequent sequencing of the 1.8 Kb fragmentcomprised in the previously characterized 6.5 Kb fragment it isinitially isolated from the plasmid p108/III2! and subsequently clonedinto a suitable vector.

The identity of the 1.8 Kb SalI fragment comprised in the 6.5 Kb PvuIfragment of clone pSN105/7 to the 1.8 Kb fragment cloned into theplasmid p108/1112 can be demonstrated on the basis of hybridizationexperiments.

Preferred within the scope of the present invention is an M13mp18 or anM13mp19 vector, each of which can be obtained commercially, for examplefrom GIBCO-BRL (Basel, CH). The cloning into these vectors and thesubsequent transfection of a suitable E. coli host can be carried out bythe methods described by Maniatis et al (1989) page 4.35-4.38!.

The recombinant phage plaques (colorless) obtained in this way can beisolated as described by Maniatis et al page 4.25; (1989)! andreplicated in a suitable E. coli host. Analysis of the recombinantphages for an insert fragment of the correct size can likewise becarried out by means of known methods using gel electrophoresis Maniatiset al page 4.39-4.40; (1989)!. A larger amount of the single-strandedDNA from the phages with the required insert is then isolated Maniatiset al page 4.29-4.30; (1989)!.

The DNA sequencing can preferably be carried out using a commercialsequencing kit. Preferred within the scope of this invention is the T7sequencing kit from PHARMACIA, which operates by the dideoxy method ofSanger (1977). This entails employing the previously isolatedsingle-stranded DNA as a template. All the sequencing operations arecarried out in accordance with the instructions of the Pharmacia T7sequencing kit (1991 instructions, No. xy-010-00-08) using the universalprimer contained in the kit and the ³⁵ S dATP which (Amersham) is usedfor the radioactive labeling. The samples obtainable in this way aresubsequently fractionated by electrophoresis on a denaturingpolyacrylamide gel (6%) and examined by autoradiography with an X-rayfilm Kodak X-OmatS!. Details of the procedure can be found in thefollowing examples and the instructions of Maniatis et al page13.45-13.58; (1989)!.

It is possible on the basis of the DNA sequence information obtainablein this way to synthesize oligonucleotides which can then be employed asprimers for sequencing the regions of the 1.8 Kb SalI fragments whichare located further inside.

NON-LIMITING EXEMPLARY EMBODIMENTS

General recombinant DNA techniques

Since many of the recombinant DNA techniques used in this invention areroutine for the person skilled in the art, a brief description of thesegenerally used techniques is to be given hereinafter. All these methodsare described in the Maniatis et al (1982) reference unless specialreference is made thereto.

A. Cutting with restriction endonucleases

The reaction mixture typically contains about 50 to 500 μg/ml DNA in thebuffer solution recommended by the manufacturer, primarily New EnglandBiolabs, Beverly, Mass. and Bohringer, Mannheim (FRG). 2 to 5 units ofrestriction endonucleases are added for each μg of DNA, and the reactionmixture is incubated at the temperature recommended by the manufacturerfor one to three hours. The reaction is stopped by heating at 65° C. for10 minutes or by extraction with phenol, followed by precipitation ofthe DNA with ethanol. This technique is also described on pages 104 to106 of the Maniatis et al (1982) reference.

B. Treatment of the DNA with polymerase in order to generate blunt ends

50 to 500 μg/ml DNA fragments are added to a reaction mixture in thebuffer recommended by the manufacturer, primarily New England Biolabs,Beverly, Mass. and Bohringer, Mannheim (FRG). The reaction mixturecontains all four deoxynucleotide triphosphates in concentrations of 0.2mM. The reaction takes place at 15° C. for 30 minutes and is thenstopped by heating at 65° C. for 10 minutes. The large fragment, orKlenow fragment, of DNA polymerase is used for fragments obtained bycutting with restriction endonucleases which generate 5'-protrudingends, such as EcoRI and BamHI. T4 DNA polymerase is used for fragmentsobtained by endonucleases which generate 3'-protruding ends, such asPstI and SacI. The use of these two enzymes is described on pages 113 to121 of the Maniatis et al (1982) reference.

C. Agarose gel electrophoresis and purification of DNA fragments fromgels

The agarose gel electrophoresis is carried out in a horizontal apparatusas described on pages 150 to 163 of the Maniatis et at. reference. Thebuffer used is the tris-acetate buffer described therein. The DNAfragments are stained by 0.5 μg/ml ethidium bromide which is normallyalready present in the gel during the electrophoresis or is added afterthe electrophoresis. The DNA is visualized by illumination withlong-wavelength ultraviolet light.

When the fragments are to be removed from the gel, an agarose which gelsat low temperature and can be obtained from Sigma Chemical, St. Louis,Mo., is used. After the electrophoresis, the required fragment is cutout, placed in a plastic tube, heated at 65° C. for about 15 minutes,extracted three times with phenol and precipitated twice with ethanol.This method is a slight modification of that described on page 170 ofManiatis et al (1982). Alternatively, the DNA can be isolated from theagarose with the aid of the Geneclean kit (Bio 101 Inc., La Jolla,Calif., USA).

D. Deletion of 5'-terminal phosphates frm DNA fragments

During the plasmid cloning steps, treatment of the vector plasmid withphosphatase reduces the recircularization of the vector (discussed onpage 13 of the Maniatis et al reference). After the DNA has been cutwith the correct restriction endonuclease, one unit of alkalinephosphatase from calf intestine, which was obtained fromBoehringer-Mannheim, Mannheim, is added. The DNA is incubated at 37° C.for one hour and subsequently twice extracted with phenol andprecipitated with ethanol.

E. Linkage of the DNA fragments

When it is intended to link together fragments with complementarycohesive ends, about 100 ng of each fragment are incubated in a reactionmixture of 20 to 40 μl with about 0.2 units of T4 DNA ligase from NewEngland Biolabs in the buffer recommended by the manufacturer. Theincubation is carried out at 15° C. for 1 to 20 hours. When it isintended to link DNA fragments with blunt ends, they are incubated asabove except that the amount of T4 DNA ligase is increased from 2 to 4units.

F. Transformation of DNA into E. coli

The E. coli strains HB101, W3101 and ED8767 are used for mostexperiments. DNA is inserted into E. coli by the calcium chloride methodas described by Maniatis et al (1982), pages 250 to 251.

G. Screening of E. coli for plasmids

After the transformation, the resulting colonies E. coli are tested forthe presence of the required plasmid by a rapid plasmid isolationmethod. Two customary methods are described on pages 366 to 369 of theManiatis et al (1982) reference.

H. Isolation of plasmid DNA on a large scale

Methods for isolating plasmids from E. coli on a large scale aredescribed on pages 88 to 94 of the Maniatis et al (1982) reference.

FIGURES

FIG. 1 FIG. 1! shows a restriction map of the 6.4 (6.5) Kb Pvul fragmentof the cosmid p98/1. The bar indicates the 1.8 kb SalI fragment and thestriped region of the bar represents the sequenced SalI-BgllIIsubfragment.

FIG. 2 FIG. 2! shows the restriction map of the 40 Kb insert of cosmidp98/1. The bars indicate the position of the four Pvul fragmentsanalyzed by gene disruption. Further Pvul sites outside of these Pvulfragments are not shown.

EXAMPLE 1

Cloning of a fragment from the `soraphen gene duster` from Sorangiumcellulosum

1.1 Construction of a cosmid gene bank from Sorangium cellulosum

1.1.1 Isolation of high molecular weight genomic DNA

The high molecular weight about 100 Kb! genomic complete DNA ofSorangium cellulosum is isolated from the strain M15 with the aid ofmethods known per se.

This specifically entails initially Sorangium cellulosum cells beingcultivated at a temperature of 28° C. in 200 ml of a G51b medium seesection: Media and buffers! with continuous agitation at 180 rpm for10,days. The cells are subsequently removed by centrifugation at 10,000rpm Sorvall GSA Rotor; DuPont Instruments, Newton, Conn., USA! andresuspended in 36 ml of an STE buffer see section: Media and buffers! towhich lysozyme Boehringer Mannheim, Mannheim, FRG! is added in a finalconcentration of 5 mg/ml. This mixture is incubated at 37° C. for 30minutes. Subsequently, 16 ml of a lysing buffer section: Media andbuffers! are added and this new mixture is incubated at 55° C. for afurther hour.

The cell lysate obtainable in this way is enriched with CsCI to a finalconcentration of 1 g/ml and this mixture is then centrifuged at 10,000rpm Sorvall SA600 Rotor; DuPont Instruments! for 10 minutes to removeparticulate constituents. A further centrifugation of the lysate iscarried out at 45,000 rpm Beckman VTi50 Rotor; Beckman Instruments Inc.,Palo Alto, USA! for a period of 18 hours and at a temperature of 20° C.

The genomic DNA can then be isolated from the readily visible, highviscosity fraction by, for example, simply puncturing the plasticcentrifuge tube with the aid of a 16 gauge injection needle and allowingthe contents to run out. The high viscosity fractions which comprisehigh molecular DNA are collected and subsequently combined.

This DNA-containing fraction is subsequently dialyzed extensivelyagainst TE buffer see section: Media and buffers! and the DNA isconcentrated by ethanol precipitation. The precipitate is centrifugedand the pellet is resuspended in 2 ml of TE buffer and stored at 40° C.until processed further.

1.1.2 Partial digestion of the high molecular weight DNA

The previously isolated DNA is subsequently digested with the aid ofsuitable restriction enzymes. In order to achieve different restrictionlevels therein, the DNA is subjected to three mutually independentpartial digestions with Sau3A Promega, Madison, Wis., USA! in a buffersolution also supplied by the manufacturer. The amount of DNA employedis in each case 100 μg, and the enzyme concentration is 0.04, 0.02 or0.01 units/μg of DNA. The incubation time is 30 minutes at 37° C. Theenzyme activity is stopped by adding Na₂ EDTA to a final concentrationof 50 mM. This reaction mixture is extracted twice withphenol:chloroform 50:50! and once with chloroform, followed by anethanol precipitation. The partially digested DNA is resuspended in TEbuffer, and all samples are pooled and adjusted to a concentration of 1mg/ml.

The DNA pretreated in this way is then heated at 65° C. for 5 minutesand fractionated according to size by centrifugation Beckmann SW-28Rotor; 25,000 rpm (82,700×g) at 20° C. for 16-18 hours! on a 10% to 40%strength sucrose density gradient 10%-40% sucrose in 5% steps in 1MNaCl, 20 mM tris pH 8.0!, 5 mM EDTA!. The gradients are fractionated inaliquots each of 0.5 ml, and samples of the order of 30 μl in size areremoved from every second aliquot, dialyzed and analyzed together with asize standard on a 0.4% agarose gel.

1.1.3 Construction of a genomic gene bank

The cosmid vector pHC79 Hohn B and Collins J, 1980! is completeddigested with BamHI, extracted with phenol:chloroform 50:50! andsubsequently precipitated and concentrated with ethanol.

Those fractions of the Sau3A partially digested S. cellulosum DNA whichcomprise DNA fragments with sizes of the order of 35 Kb to 45 Kb arepooled and concentrated via an ethanol precipitation.

This is followed by treatment with alkaline phosphatase from calfstomach Promega!, which is carried out in accordance with themanufacturer's instructions.

The ligation of the cosmid DNA to the S. cellulosum DNA which has beenfractionated according to size is carried out with the aid of a T4 DNAligase. This entails the two DNA starting materials being mixed togetherin approximately equimolar amounts about 6 μg of S. cellulosum DNA andabout 1 μg of vector DNA!, followed by a heat treatment at 65° C. for 15minutes and by a renewed ethanol precipitation. The final concentrationof the complete DNA is 500 μg of DNA/ml to 800 μg of DNA/ml in a totalvolume of 10 μl. This reaction mixture is incubated first at roomtemperature for several hours and to follow at 15° C. for a further 10to 14 hours.

One to two μl of the ligation mixture are then packaged in lambda phagesusing an in vitro commercial packaging kit which can be obtained, forexample, from STRATAGENE, Inc. in La Jolla, Calif. (USA) or PROMEGA inMadison, Wis., USA. The resulting phage particles are used for infectionof bacteria of a suitable recA⁻ E. coli strain for example of the E.coli strain HB 101!. The specific procedure for this is such that

(a) the bacteria are first cultivated as overnight culture in a Luriabroth medium 10 g/l Bacto tryptone; 5 g/l yeast extract; 5 g/l NaCl!supplemented with 0.4% maltose and 10 mM MgSO₄ ;

(b) the culture is diluted in the same medium and left to grow until anoptical density OD₆₀₀ ! of 0.5 mid-log phase! is reached about 2 hours!;

(c) 0.2 ml of the E. coli culture is transferred into a sterileEppendorf tube, 50 μl of the "packaged" phage particles are added, andthe complete mixture is thoroughly mixed;

(d) after incubation at 37° C. for 15 minutes, 1 ml of Luria brothsolution is added, and the incubation is continued for a further hour;

(e) to select transfected clones, aliquots of 0.1 ml are plated out on aLuria broth agar supplemented with 50 mg/l ampicillin; and

(f) plasmid DNA is isolated with the aid of known methods from thecolonies resulting after selection.

Titration of the phage material reveals a total of about 50,000 phageparticles/ml.

1.2 Preparation of the radioactively labelled DNA probe from S.violacerouber Tu22

Used as starting material for preparing the DNA probe is the plasmidplJ5200 Sherman et al, 1989! which comprises a 4.6 Kb BamHI fragment ofthe graI region granaticin gene cluster ORF 1-4! of Streptomycesviolaceoruber Tu22 cloned into the BamHI cleavage site of pUC18obtainable from GIBCO BRL, Basel, CH!.

In order to obtain a DNA fragment which is free of DNA sequences whichhave homologies with corresponding pBR322 or pUC18 sequences, the 4.6 KbBamHI fragment is first cloned into the streptomyces vector pIJ486 Wardet al, 1986!. For this, about 3 μg of the plasmid pIJ5200 Sherman et al,1989! are completely cut with BamHI, and the resulting fragments areseparated from one another by agarose gel electrophoresis.

The required 4.6 kb fragment is then removed from the agarose byelectroelution. For this, it is first cut out as an approximately 2 mmto 3 mm wide agar strip from the agarose gel with a sterile scalpel andplaced in a dialysis tube prepared dialysis tubing 1/4 inch diameterobtainable, for example, from BRL!. The dialysis tube containing theagarose block is then treated in 1×TAE buffer iris-acetate 0.04M; EDTA0.002M! in a horizontal agarose electrophoresis chamber with a currentof 100 milliampere so that the current flows at right angles to the longaxis of the dialysis tube for 2 hours. After reversal of the current for45 seconds, the dialysis tube is opened and the contents (buffer andagarose block) are forced, using a 5 ml syringe, slowly through a glassfiber filter Whatman GF/C; Whatman Scientific Ltd., Kent, GB!. Thefilter is then washed with 200 μl of TAE buffer 1×!, and then tRNAtransfer RNA from baker's yeast, obtainable, for example, from BohringerMannheim, FRG! is added to a final concentration of 5 μl/ml to thefiltered sample. The DNA sample is then extracted with phenol/chloroformManiatis et al, 1982, page 458! and precipitated with ethanol Maniatiset al, 1982; page 461!. The resulting precipitate is taken up in 30 μlto 40 μl of TE buffer.

Approximately 0.5 μg of the fragment obtained in this way is mixed withabout 0.1 μg of pIJ486 Ward et al, 1986! vector DNA which is previouslycut with BamHI, and is precipitated with ethanol Maniatis et al, 1982;page 461! and ligated with T4 DNA ligase obtainable from BohringerMannheim, FRG! in 20 μl of reaction buffer as specified by manufacturer!at about 15° C. overnight. 2-5 μl of this ligation mixture aretransformed into the Streptomyces pilosus strain M1/5 Schupp et al,1988! by the protoplast method described by Hopwood et al, (1985)!. Thetransformants are selected after incubation (28° C.) for 18 hours bypouring 4 ml of soft nutrient agar (Difco) which contains 250 μg/mlthiostrepton see Schupp et al, 1985! onto the R2YE agar Hopwood et al,1988!. After incubation at 28° C. for 7 days, colonies are selected forisolation of plasmid DNA. The DNA isolation is carried out by a slightmodification according to Schupp et al, 1988! of the method of Birnboimand Doly (1979). The isolated plasmid DNA is subsequently digested withBamHI, and both digested and undigested DNA is analyzed with the aid ofgel electrophoresis.

The colonies which possessed an additional BamHI fragment of 4.6 Kb arecultivated in 500 ml of a 148G medium Schupp et al, 1986!, and theplasmid DNA is isolated with the aid of the CsCl gradient methodsHopwood et al, 1985!. The plasmids obtainable in the manner describedpreviously and having the 4.6 Kb BamHI fragment of S. violaceoruber arecalled p82/11 and p82/21.

For radioactive labeling of the 4.6 Kb DNA fragment, about 1 ug of theplasmid p82/21 is completely cut with BamHI, and the fragments obtainedin this way are separated from one another by agarose gelelectrophoresis. The 4.6 Kb fragment is subsequently isolated from theagarose gel by electroelution (see above). Approximately 0.2 μg of the4.6 Kb BamHI fragment isolated in this way is employed for theradioactive labeling by means of a nick translation Rigby D. W. J. etal, 1977!. The nick translation can be carried out using the nicktranslation's system commercially available from GIBCO BRL (BethesdaResearch Laboratories Life Technologies Inc.) in Basle, CH, with thelabeling taking place, according to the manufacturer's instructions,with 80 ZμCi of d-CTP ³² P. The labelled 4.6 Kb fragment can beseparated from the non-incorporated nucleotides by passage through anick ™column containing Sephadex G-50 Pharmacia Biosystems, Dubendorf,CH! and subsequent elution of 200 μl fractions with TE buffer.

1.3 Hybridization of individual clones within the cosmid gene bank ofSorangium-cellulosum using the DNA probe from S. violaceoruber

It is possible by infection of bacteria of the E. coli strain HB101 with100 μl of in vitro packaged lambda phages according to Example 1.1!,after plating on Petri dishes with LB agar supplemented with ampicillin60 μg/ml!, to obtain a large number up to 1300 colonies and more! ofampicillin-resistant colonies. These colonies can be tested by means ofcolony hybridization on nylon filters in the following way Amershaminstructions for Hybond filters!:

A nylon filter Amersham Transfer Membrane Hybond-N; AmershamInternational, Amersham, UK! is placed on the colonies, and after oneminute, --with the colonies upwards--, placed on a sterile filter paperWhatman No.3!. To produce an imprint (replica) of the colonies, a secondfilter is pressed onto the first and subsequently placed on LB agarplates supplemented with 60 μg/ml ampicillin! and incubated thereon at37° C. until the colonies have a diameter of about 1 mm. The firstfilter is then, with the colonies upwards, placed on a stack of a totalof 5 Whatman No.3 papers impregnated with a denaturation solution 1.5MNaCl; 0.5M NaOH!. After acting for 7 minutes, the filters are placed inthe same way on a new filter impregnated this time with a neutralizationsolution 1.5M NaCl; 0.5M Tris-HCl (pH 7.2); 0.001M EDTA!. This solutionis allowed to act for 3 minutes before the treatment with theneutralization solution is repeated once more.

The filters are then briefly immersed in 2×SSC 0.3M NaCl; 0.03M sodiumcitrate (Maniatis et al, 1982! and, for drying, placed on dry WhatmanNo.3 filters (colonies upwards) and dried in air. In order to fix theDNA, the filters are wrapped in Saran film Dow Chemical Company! andirradiated with UV light (312 nm) in a UV transilluminator CAMAGReprostar, Camag, Muttenz, CH! for 5 minutes (colonies downwards).

Subsequently, for the prehybridization, the filters are incubated at 65°C. for 90 min with a buffer of the following composition: 2×SSC Maniatiset al, 1982+1×Denhard Maniatis et al, 1982!+0.5% SDS+50 μg/ml salmonsperm DNA DNA sodium salt, Type III, from Salmon Testes; SIGMA ChemicalCo., St. Louis, USA! (denatured by heating at 100° C. for 10 min.) Forthe hybridization, about 2×10⁵ cpm/ml of the 4.6 Kb DNA fragment,labelled by nick translation, from plasmid pCT82/21 see Example 1.2!,which has previously been denatured by heating at 100° C. for 10 min.,are added.

The actual hybridization then takes place at 65° C. for 24 hours.

After the hybridization, the filter is washed first in 2×SSC at roomtemperature for 15 min, then in 2×SSC at 60° C. for 15 min, subsequentlyin 2×SSC+0.1% SDS at 60° C. for 30 min and finally in 0.5×SSC at 60° C.for 15 min. The subsequent autoradiography is carried out with X-rayfilm for example Fuji RX (medical X-ray film)! for 24 hours.

1.4 Analysis of the cosmid-containing colonies by means of plasmidisolation and Southern hybridination

The cosmid clones obtaininable by the previously described method andshowing the strongest signals in the colony hybridization aretransferred from the agar plate into 2 ml of LB medium and incubated at37° C. overnight. A 1 ml aliquot of these cultures is then used forisolating the plasmid DNA as described by Maniatis et al, (1982); pages368-369!.

The isolated plasmids are then digested with SalI, and the fragmentsobtained in this way are fractionated in a 0.8% tris-acetate agarose gel1×TAE! at 1.2 volt/cm for 15 hours. The gel is subsequently treatedfirst for 2×15 minutes in a denaturing solution 1.5M NaCI; 0.5M NaOH!and subsequently for 2×15 minutes in a neutralizing solution 1.5M NaCI;0.5M Tris-HCl; 1 mM EDTANa2 (pH 7.2)! shaking gently at roomtemperature. The DNA is then transferred by means of a Southerncapillary blotting Maniatis et al, 1982; pages 383-386! using 10-foldconcentrated SSC buffer to a nitrocellulose membrane 0.45μnitrocellulose; Bio-Rad Laboratories, Richmond, USA! and subsequentlyfixed on the membrane at 80° C. in vacuo for 2 hours.

For the subsequent hybridization, the filter is first incubated for theprehybridization at 65° C. for 120 min with a buffer of the followingcomposition: 2×SSC (Maniatis et al, 1982) +1×Denhard (Maniatis et al,1982)+0.5% SDS+50 μg/ml salmon sperm DNA (denatured by heating at 100°C. for 10 min.). For the actual hybridization, about 8×10⁶ cpm of the4.6 Kb DNA fragment labelled by nick translation from the plasmid p82/21see Example 1.2!, which is previously denatured by heating at 100° C.for 10 min, are added. The hybridization then took place at 65° C. for36 hours.

After the hybridization, the filter is washed twice in 2×SSC+0.1% SDS at55° C. for 20 min and then in 0.2×SSC+0.1% SDS at the same temperaturefor 20 min. The subsequent autoradiography is carried out by means of anX-ray film for example Fuji RX (medical X-ray film)! for 48 hours.

A cosmid p98/1)! which shows a strong band of 1.8 Kb in theautoradiograph is selected for further work.

1.5 Subcloning of the 1.8 kb SalI fragment from cosmid p98/1 into the E.coli vector pBR322

For preparation of a larger amount of the cosmid p98/1, a 20 ml cultureof the corresponding E. coli strain HB101/p98/1 see Example 1.1! isprepared in an LB medium and incubated overnight. It is then possible toisolate a larger amount of DNA of cosmid p98/1 from this culture 15 ml!see Maniatis et al, (1982); pages 368-369!.

About 3 μg of the cosmid p98/1 DNA are completed digested with SalI, andthe fragments obtainable in this way are fractionated by electrophoresison a 0.8% tris-acetate agarose gel 1×TAE! at 1.2 volt/cm for 15 hours.The required 1.8 Kb DNA fragment is then removed from the agarose byelectroelution in analogy to the procedure described in Example 1.2.

30 μl of the sample of the 1.8 Kb SalI fragment obtained in this way aremixed with about 0.05 μg of the plasmid pBR322 which has previously beencut with SalI, and are precipitated with ethanol Maniatis et al, (1982);page 461! and then ligated with T4 DNA ligase Bohringer Mannheim, FRG!in 20 μl of reaction buffer as specified by manufacturer! at about 15°C. overnight.

The resulting ligated DNA 5 μl! is subsequently cloned into cells of theE. coli strain HB101 which have been made competent as described byManiatis et al (1982) pages 250-251! and plated out on LB agar which issupplemented with 50 μg/ml ampicillin.

Tetracycline-sensitive colonies can be found by differential screeningof the resulting transformed colonies on LB agar with 50 μg/mlampicillin or with 50 μg/ml ampicillin and 20 μg/ml tetracycline, andtheir plasmid DNA can be isolated as described by Maniatis et al, 1982;pages 368-369!. The isolated plasmid DNA is then cut with SalI andanalyzed by agarose gel electrophoresis for the size of its insertedfragments, employing as comparison the cosmid p98/1 which is alsoapplied.

A plasmid p102/III2! which comprises an additional fragment of therequired size can then be isolated from the gel in the manner previouslydescribed. The identity of this additional fragment to the 1.8 Kb SalIfragment from the cosmid p98/1 can then be confirmed in a Southerntransfer as described in Example 1.4! and hybridization with the 4.6 KbBamHI DNA probe from S. violaceoruber.

1.6 Sequencing of a 0.78 Kb SalI/BglII subfragment of the 1.8 Kb SalIfragment of plasmid p108/III2.

For the sequencing, the 1.8 kb SalI fragment is first isolated from theplasmid p108/III2 described in Example 1.5. For this, about 2 μg of theplasmid p108/III2 are completely cut with SalI and fractionated byelectrophoresis in a 0.8% tris-acetate agarose gel at 1.2 volt/cm for 15hours. The 1.8 kb SalI fragment is then isolated by electroelution inanalogy to the procedure described in Example 1.5.

The SalI fragment about 0.3 μg) can subsequently be ligated directlyinto the SalI cleavage site of the vector M13mp18 about 0.02 μg!.

As an alternative to this, the SalI fragment can also be additionallycut with BglII and ligated in the form of two BglII-SalI subfragments 2fragments, total about 0.3 μg! into the vector M13mp19 which ispreviously cut with SalI and BamHI about 0.1 μg! the ligation with T4DNA ligase can be carried out as described in Example 1.5!. The M13mp18and M13mp19 vectors are commercially available from GIBCO-BRL (Basle,CH). The cloning into these vectors and the subsequent transfection ofE. coli JM101 cells can be carried out by the method described byManiatis et al (1989) page 4.35-4.38!.

In each case 12 of the recombinant phage plaques (colorless) obtained inthis way are isolated as described by Maniatis et al page 4.25; (1989)!and replicated in E. coli JM101. Analysis of the recombinant phages foran insert fragment of the correct size can likewise be carried out bymeans of known methods using gel electrophoresis Maniatis et al page4.39-4.40; (1989)!. A larger amount of the single-stranded DNA from thephages with the required insert is then isolated Maniatis et al page4.29-4.30; (1989)!.

The DNA sequencing can be carried out with the aid of the T7 sequencingkit from Pharmacia by the dideoxy method of Sanger (1977). For this, thepreviously isolated single-stranded DNA is employed as template. Allsequencing operations are carried out in accordance with theinstructions of the Pharmacia T7 sequencing kit (1991 instructions, No.xy-010-00-08) using the universal primer contained in the kit and the ³⁵S dATP Amersham International; Amersham, UK! which is used for theradioactive labeling. The samples obtainable in this way aresubsequently fractionated by electrophoresis on a denaturingpolyacrylamide gel (6%) and examined by autoradiography with an X-rayfilm Kodak X-OmatS!. Details of the procedure can be found in theinstructions of Maniatis et al page 13.45-13.58; (1989)!.

4 oligonucleotides are synthesized on the basis of the DNA sequenceinformation obtained above in order to determine the DNA sequence in thecentral region of the two SalI-BglII subfragments.

The oligonucleotides Oli1 and Oli2 can be employed as primers for thesequencing in the following sequencing strategy to sequence the5'-located SalI/BglII subfragment. The 2 oligonucleotides have thefollowing base sequence:

    ______________________________________                                        5' TAC TGG TAT CGA AAC CT 3'                                                                    (151-167) = Oli1 (SEQ ID NO: 2)                             5' GAA GAC GAC GTG GTC TT 3'                                                                    (642-626) = Oli2 (SEQ ID NO: 3)                             ______________________________________                                    

The sequencing strategy for the 0.78 Kb SalI/BglII fragment can bedepicted diagrammatically as follows: ##STR1## 2. Subcloning of PvuIfragments of the cosmid p98/1 and conjuative transfer into S. cellulosum

Since the vector pSUP2021 which is used for the gene disruptionexperiment described in Example 3 has a unique PvuI cleavage site in theAmp^(R) gene, PvuI fragments are correspondingly used hereinafter forthe subcloning steps and the conjugative transfer.

2.1 Subcloning of PvuI fragments of the cosmid p98/1 into the plasmidpSUP2021

Cosmid p98/1 about 4 μg! is completely cut with PvuI, and the resultingfragments are fractionated by electrophoresis on a 0.8% tris-acetateagarose gel 1×TAE buffer! at 1.2 volt/cm for 18 hours (FIG. 2) inanalogy to the procedure described in Example 1.5.

The well-separated fragments about 6.5 Kb, about 10 Kb and a fraction of4 Kb and 4.2 Kb! are isolated by electroelution by the method describedin Example 1.5 and each resuspended in 40 μl of TE buffer. 35 μl of eachof the samples obtainable in this way of the 4 fragments are mixed withabout 0.05 μg of the plasmid pSUP2021 Simon R et al, 1983! which ispreviously cut with PvuI, precipitated with ethanol as described byManiatis et al (1982); page 461! and subsequently ligated with T4 DNAligase obtainable from Bohringer, Mannheim, FRG! in 20 μl of reactionbuffer at about 15° C. overnight.

E. coli HB101 cells made competent as described by Maniatis et al (1982)are transformed Maniatis et al (1982); page 250-251! with in each case10 μl of the 3 DNA samples obtainable from the ligase reaction describedabove, and plated out on LB agar with 30 μg/ml chloramphenicol. Each ofthe colonies transformed in this way is tested for its ampicillinsensitivity. The ampicillin-sensitive colonies are subsequentlyidentified and used as starting material for isolating the plasmid DNAManiatis et al (1982); pages 368-369!. The isolated plasmids are thencut with Pvul and analyzed by an agarose gel electrophoresis. Cosmidp98/1 cut with PvuI is used as comparison standard. It is possible inthis way to identify a total of three plasmids which comprise therequired fragment. The plasmid with the fragment comprising about 6.5 Kbis called pSN 105/7. The plasmid with the fragment comprising about 10Kb is called pSN120/10. The plasmids with the fragments comprising 4 Kband 4.2 Kb are called pSN120/43-49 and pSN120/46 respectively containedfragments 4 and 4.2 kb in size.

An exact measurement of the above mentioned fragments turns out thefollowing sizes:

    ______________________________________                                        size used in                                                                            exact size as    plasmid                                            the text  Kb!                                                                           measured  Kb!    name                                               ______________________________________                                        10        12.5             pSN120/10                                          6.5       6.4              pSN105/7                                           4.2       4                pSN120/43-49                                       4         3.8              pSN120/46                                          ______________________________________                                    

2.2 Conjugal transfer of recombinant plasmids into S. cellulosum

2.2.1 Transformation of plasmids pSN105/7, pSN120/10, pSN120/43-49 andpSN120/46 into E. coli

The plasmids pSN105/7, pSN120/10, pSN120/43-49 and pSN120/46, whosepreparation and isolation has previously been described in Example 7.1,are transformed into the E. coli strain ED8767 which comprises thehelper plasmid pUZ8 Hedges R. W. and Matthew M (1979)!.

The plasmid pUZ8 is a derivative of the plasmid RP4 which comprises awide host range and which is described by Datta et al (1971). Themodifications compared with the initial RP4 plasmid essentially relateto the ampicillin-resistance gene and to the insertion element IS21,both of which are deleted, and to the incorporation of an additionalgene which confers resistance to mercury ions see Jaoua et al (1987)!.

The colonies resulting after transformation and subsequent incubation onLB agar supplemented with tetracycline 10 μg/ml! and chloramphenicol 25μg/ml! for 24 hours are subjected to a differential screening byparallel plating out on ampicillin-containing 60 μg/ml! andampicillin-free medium. It is subsequently possible to isolate thosecolonies which, because of the integration of the Sorangium DNAfragments, have lost their ampicillin resistance. The culturesobtainable in this way can then be employed directly as donor cells forthe conjugative transfer of the recombinant plasmids into Sorangiumcellulosum cells.

2.2.2 Conjugative transfer

For the actual transfer, 15 ml of a Sorangium cellulosum SJ3 culture inthe stationary phase 1-4×10⁹ cells/ml! are mixed with 10 ml of alate-log phase culture of E. coli ED8767 donor cells which comprise acomparable number of cells. These are then centrifuged together at 4000rpm for 10 minutes and resuspended in 500 μl of a G51b or G51t medium.

It proves advantageous in this case to expose the Sorangium recipientcells to a brief heat treatment in a water bath before the conjugationwith E. coli. The best transfer results with the Sorangium cellulosumstrain SJ3 can be achieved with a heat treatment at a temperature of 50°C. for 10 minutes. Transfer frequencies of 1-5×10⁻⁵ can be achievedunder these conditions, which corresponds to an increase by a factor of10 compared with a method without previous heat treatment.

Transfer to plates with So1E solid medium is followed by incubation at30° C. for two days. The cells are then harvested and resuspended in 1ml of G51b or G51t medium. 100 μl of this bacterial suspension areplated out on a selective So1E medium which, besides kanamycin 25 mg/l!,also contains phleomycin 20 to 35 mg/l! and streptomycin 300 mg/l! asselective agents. The counter-selection of the donor strains E. coliW3101 (pME305)! takes place with the aid of streptomycin.

The colonies which have grown on this selective So1E medium after anincubation time of 10 to 14 days at a temperature of 30° C. aretransconjugants of Sorangium cellulosum which have acquired phleomycinresistance by conjugative transfer of the plasmids pSN105/7, pSN120/10,pSN120/43-49 and pSN120/46. These phleomycin-resistant colonies can beused for the subsequent molecular biological investigations. Thetransformation frequency for the transfer of the plasmid DNA toSorangium averages 1-3×10⁻⁶ based on the SJ3 recipient strain.

3. Characterization of the previously identified fragments

3.1 Assignment of the 1.8 Kb SalI fragment into the 6.5 Kb PvuI fragmentof cosmid p98.1

The plasmids pSN105/7, pSN120/10, pSN120/43-49 and pSN120/46, whosepreparation and isolation has been described in Example 2.1, arecompletely digested with SalI and PvuI, and the fragments arefractionated on a 0.8% tris-acetate agarose gel as in Example 1.4 andtransferred by Southern capillary blotting onto a nitrocellulosemembrane. The hybridization is carried out using the 1.8 Kb SalIfragment of plasmid p108/III2 as probe, under the conditions describedin Example 1.4, carrying out the last filter washing step at 65° C. (in0.2×SSC+0.1% SDS).

Evaluation of the agarose gel and of the autoradiograph shows that theclone pSN105/7 comprises in its PvuI fragment which comprises 6.5 Kb a1.8 Kb SalI fragment which is identical to the 1.8 Kb fragment clonedinto the plasmid p108/112.

3.2 Characterization of the 6.5 Kb PvuI fragment of cosmid p98/1 byrestriction cleavage sites

The plasmid pSN105/7 is digested with PvuI as described in Example 3.1,and the two fragments obtained in this way are fractionated on a 0.8%tri-acetate agaros gel 1×TAE! at 1.2 volt/cm for 15 hours. The DNAfragment which is 6.5 Kb in size is then removed from the gel byelectroelution in analogy to the procedure described in Example 1.2 andtaken up in 30 μl of TE buffer.

To characterize the 6.5 Kb PvuI fragment by restriction cleavage sites,the resulting 6.5 Kb DNA fragment is digested with, in each caseindependently of one another, BglII, SphI and SmaI as well ascompletely, and the size of the resulting fragments is determined afterfractionation on a tris-acetate agarose gel 1×TAG!. For this,HindIII-cut λ DNA (obtainable from GIBCO-BRL, Basle, Switzerland) isemployed as standard for comparison of sizes of the DNA fragments on thesame gel. In order to be able to establish unambiguously the position ofthe individual fragments with respect to one another, the 6.5 Kbfragment is additionally digested with all conceivable combination oftwo of the abovementioned restriction enzymes in each case.

Digestion with BglII yields two fragments 4.3 Kb and 2.3 Kb in size, andthat with SphI yields 4 fragments 2.8 Kb, 1.5 Kb, 1.5 Kb and 0.7 Kb insize. Digestion with SmaI yields 3 fragments 2.9 Kb, 2.0 Kb and 1.6 Kbin size. In this case the actual size of the fragments may differ by±10% from the stated value because of the method of measurement used.The position of the BglII and SphI cleavage sites on the 6.5 Kb fragmentof cosmid p98/1 can be established on the basis of the fragment sizesfound as depicted in FIG. 1.

4. Function test

4.1 Analysis of the effect of the PvuI fragment of the cosmid p98/1 onthe soraphen production by S. cellulosum after gene disruption

Transfer of plasmids comprise DNA sections which are homologous withcorresponding sections within the Sorangium cellulosum genome leads tointegration of said DNA sections into the chromosomal Sorangiumcellulosum DNA at the site of the homology via homologous recombination.

If the homologous region is a section within a gene cluster, for examplethat for soraphen biosynthesis, the plasmid integration leads toinactivation of this cluster by so-called gene disruption. This methodthus allows the function of a cloned DNA fragment in Sorangiumcellulosum to be analyzed.

To check the function of the PvuI fragments of cosmid p98/1, well-grown,transconjugant colonies see Example 2.2.2! are removed from the masterplates with a sterile plastic loop and streaked on a selective So1Emedium which contains kanamycin 25 mg/l!, phleomycin 20-35 mg/l! andstreptomycin 300 mg/l! as selective agents. After an incubation time at30° C.! of 8 10 days, about 1-2×10⁹ cells are removed with a sterileplastic loop and transferred into 2 ml of G-55 medium without resin!.

After incubation at 30° C. and 180 rpm for 3-4 days, samples each of 1ml 1×4×10⁹ cells/ml! are transferred into an Erlenmeyer flask with 10 mlof G55 medium without resin!. After an incubation under identicalconditions for a further 3-4 days, another transfer is carried out 5 ml;1-4×10⁹ cells/ml! but this time into a G55 medium 50 ml! whichadditionally contains an adsorber resin such as, for example, AMBERLITEXAD-1180® ROHM & HAAS DEUTSCHLAND GmbH, Frankfurt, FRG!. Quantitativedetermination of the soraphen produced takes place after incubation at30° C. and 180 rpm for 7 days.

The soraphen determination is carried out with the aid of HPLC analysis.This entails the cultures initially being filtered with suction througha polyester filter Sartorius, B 420-47-N!. The resin remaining on thefilter is then resuspended in 50 ml of isopropanol and extracted at 30°C. and 180 rpm for one hour. 1 ml is removed from this suspension andcentrifuged at 12,000 rpm in an Eppendort Microfuge. 200 μl of thesupernatant are diluted with 600 μl of isopropanol, and the amount ofsoraphen contained therein is determined by means of an HPLC with UVdetector. The detection wavelength is at 210 nm.

In total, 35 different transconjugants with integrated pSN105/7 plasmid,14 transconjugants with pSn120/10 plasmid, 18 transconjugants withpSN120/43-49 plasmid and 8 transconjugants with pSN120/46 plasmid weretested in the manner described previously.

HPLC analysis revealed in this case that all the transconjugants nolonger produce soraphen. By contrast, soraphen A was detectable in aconcentration of 50-100 mg/l with the positive controls, with the SJ3recipient strain and with the transconjugant strain with the recombinantplasmid pSJB55.

This means that, after integration into the chromosomal Sorangiumcellulosum DNA, all 4 PvuI fragments unambiguously block soraphenproduction. These fragments thus comprise a part of the genes which areinvolved directly or indirectly in soraphen synthesis.

5. Identification of the `soraphen gene duster`

5.1 Isolation of cosmid clones with overlapping inserts

Clones which yield with fragments of the insert of p98/1 a stronghybridization signal are selected with the aid of colony hybridizationaccording to the method described under 1.3 from the cosmid gene bankproduced as in Example 1.1. Used as hybridization probe is either theradioactively labelled 1.8 Kb fragment of p98/1 or else preferablyselected SalI fragments which can be assigned on the basis of thedistribution of the SalI fragments in the sorangium part of p98/1 to theleft or right flanking zones of the insert of p98/1.

5.2 Confirmation of the overlap of novel cosmids with p98/1

It is possible by hybridization analysis to identify fragments whichoverlap the insert from p98/1 in the 3' or 5' direction. These areisolated and employed for the subsequent walking steps.

The hybridization analysis comprises the following specific steps:

The plasmid DNA is isolated by the method described in Example 1.4 fromthe cosmid clones which show a strong signal in the method describedpreviously compare 1.4!. The isolated plasmids are then digested withSalI and, in parallel, with Pvul, and the fragments obtained in this wayare fractionated on an agarose gel and subsequently transferred by meansof Southern capillary blotting Maniatis et al, 1982; pages 383-386! to anitrocellulose membrane.

It is possible by hybridization of the filter with the 1.8 Kb SalIfragment or with a SalI fragment from the flanking zone of the insert ofp98/1 as probe for the overlap of the novel cosmid clones to beconfirmed and assigned to the right or left side of the insert of p98/1.The methods mentioned here can be carried out exactly in accordance withthe techniques described under 1.4.

The number of common SalI and PvuI fragments found in the plasmid p98/1and the novel cosmid clones provides further information about the sizeof the overlaps in the insert region. It is possible, where appropriate,to isolate further cosmids with regions of the Sorangium cellulosumchromosome which are located even further left or right of the insertfragment from plasmid p98/1. For this, the above procedure is repeatedwith a SalI fragment of a "right" or "left" cosmid relative to the p98/1insert as probe.

5.3. Function analysis

The adjacent and overlapping DNA fragments identified in the mannerdescribed above are examined for their function in soraphen biosynthesiswithin the scope of a `gene disruption` as described in Examples2.1-2.2.2. The walking process with novel cosmids is continued untilfragments which have no effect on soraphen production and are thuslocated outside the `soraphen gene cluster` are reached. It is possiblein this way to locate and clone the complete gene cluster whichcomprises the insert from cosmid p98/1.

5.4. Mapping of the restriction fragments

It is possible by restriction mapping to establish the sequence of theresulting restriction fragments and thus to construct an accuraterestriction map of the complete region. In parallel with this, the DNAsequence of the individual fragments is determined by the proceduredescribed in Example 1.6.

DEPOSITION

The following microorganisms and plasmids have been deposited within thescope of the present application at the "Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH", which is recognized asinternational depository in accordance with the Budapest Treaty, inBraunschweig (FRG) in compliance with the requirements of the BudapestTreaty on the international recognition of the deposit of microorganismsfor the purposes of patent procedure.

    ______________________________________                                        Microorganism/ Date of      Deposit                                           Plasmid        deposit      number                                            ______________________________________                                        pSN105/7       25.08.1992   DSM 7217                                          cloned in E. coli                                                             ______________________________________                                    

The following microorganisms and plasmids have been deposited within thescope of the present application at the Agricultural Research CultureCollection (NRRL), 1815N. University Street, Peoria, Ill. 61604, whichis recognized as international depository in accordance with theBudapest Treaty, in compliance with the requirements of the BudapestTreaty on the international recognition of the deposit of microorganismsfor the purposes of patent procedure.

    ______________________________________                                        Microorganism/                                                                              Date of      Deposit                                            Plasmid       deposit      number                                             ______________________________________                                        p98/1 cloned in                                                                             May 20, 1994 NRRL B-21255                                       E. coli HB101                                                                 ______________________________________                                    

    ______________________________________                                        MEDIA AND BUFFER SOLUTIONS                                                    ______________________________________                                        G51b Medium (pH 7.4)                                                          Glucose                     0.2%                                              Starch                      0.5 %                                              potato starch, Noredux type; CERESTAR ITALIA S.p.a.,                         Milan, Italy!                                                                 Peptone  DIFCO Laboratories, USA!                                                                         0.2%                                              Probion S                   0.1%                                               Single Cell Protein; HOCHST AG, Frankfurt, FRG!                              CaCl.sub.2 × 2 H.sub.2 O                                                                            0.05%                                             MgSO.sub.4 × 7 H.sub.2 O                                                                            0.05%                                             HEPES  FLUKA!               1.2%                                              G51t Medium (pH 7.4)                                                          Glucose                     0.2%                                              Starch                      0.5%                                               potato starch, Noredux type; CERESTAR ITALIA S.p.a.,                         Milan, Italy!                                                                 Tryptone  MARCO, Hackensack, NJ USA!                                                                      0.2%                                              Probion S                   0.1%                                               Single Cell Protein; HOCHST AG, Frankfurt, FRG!                              CaCl.sub.2 × 2 H.sub.2 O                                                                            0.05%                                             MgSO.sub.4 × 7 H.sub.2 O                                                                            0.05%                                             HEPES  FLUKA!               1.2%                                              G52c Medium (pH 7.4)                                                          Glucose                     2.0 gl                                            Starch                      8.0 gl                                             potato starch, Noredux type; CERESTAR ITALIA S.p.a.,                         Milan, Italy!                                                                 Soybean flour deoiled       2.0 g/l                                            MUCEDOLA S.r.l., Settimo Milanese, Italy!                                    Yeast extract               2.0 g/l                                            FOULD & SPRINGER, Maison Alfort, France!                                     CaCl.sub.2 × 2 H.sub.2 O                                                                            1.0 g/l                                           MgSO.sub.4 × 7 H.sub.2 O                                                                            1.0 g/l                                           Fe-EDTA  8 g/l stock solution!                                                                            1.0 ml                                            HEPES  FLUKA!               2.0 g/l                                           Distilled water ad          1000 ml                                           pH is adjusted to 7.4 with NaOH before sterilization                           120° C. for 20 minutes!. pH after sterilization: 7.4.                 Medium: G-55 200 mlflask with 50 ml of medium                                 Starch                      8.0 g/l                                            potato starch, Noredux type; CERESTAR ITALIA S.p.a.,                         Milan, Italy!                                                                 Dextrin from potatoes  supplied by Blattmann,                                                             8.0 g/l                                           Wadenswil, CH!                                                                Glucose                     2.0 g/l                                           Soybean flour deoiled       2.0 g/l                                            MUCEDOLA S.r.l., Settimo Milanese, Italy!                                    Yeast extract               2.0 g/l                                            FOULD & SPRINGER, Maison Alfort, France!                                     DL-aspartic acid (C.sub.4 H.sub.7 NO.sub.7)                                                               1.0 g/l                                           CaCl.sub.2 × 2H.sub.2 O                                                                             1.0 g/l                                           MgSO.sub.4 × 7H.sub.2 O                                                                             1.0 g/l                                           HEPES (Fluka No. 54461)     12.0 g/l                                          Fe-EDTA (8 g/l stock solution)                                                                            1.0 g/l                                           MZM 1 (resin)               50.0 ml/l                                         Tap water ad 1000 ml                                                          pH before ster.: adjust to 7.5 with NaOH                                      pH after ster.: 7.4-7.5                                                       Inoculum: 5 ml of a well-grown culture  ca 1-4 × 10.sup.9               cells/ml!                                                                     So1E Medium (pH 7.4)                                                          Glucose*                    0.35%                                             Tryptone  MARCO, Hackensack, NJ USA!                                                                      0.05%                                             MgSO.sub.4 × 7 H.sub.2 O                                                                            0.15%                                             Ammonium sulfate*           0.05%                                             CaCl.sub.2 × 2 H.sub.2 O*                                                                           0.1%                                              K.sub.2 HPO.sub.4 *         0.006%                                            Sodium dithionite*          0.01%                                             Fe-EDTA*                    0.0008%                                           HEPES  FLUKA!               1.2%                                              Supernatant of a sterilized,                                                                              3.5% (v/v)                                        stationary S. cellulosum culture*                                             Agar                        1.5%                                              LB Medium                                                                     Tryptone                    10.0 g.l                                          Yeast extract               5.0 g/l                                           NaCl                        5.0 g/l                                           STE buffer (pH 8.0)                                                           Sucrose                     25%                                               EDTANa2                     1 mM                                              Tris-HCl                    10 mM                                             RLM buffer (pH 7.6)                                                           SDS                         5%                                                EDTANa2                     125 mM                                            Tris-HCl                    0.5 mM                                            TER buffer                                                                    Tris-HCl (pH 8.0)           10 mM                                             1 mM EDTANa2                1 mM                                              RNAse                       10 μg/ml                                       Litigation buffer                                                             MgCl.sub.2                  0.1 M                                             Tris-HCl (pH 7.8)           0.5 M                                             Lysing buffer  pH 7.6!                                                        Na-sarkosyl                 5%                                                Na.sub.2 -EDTA              125 mM                                            Tris-HCl                    0.5 M                                             TE buffer                                                                     Tris-HCl  pH 8.0!           10 mM                                             Na.sub.2 EDTA               1 mM                                              ______________________________________                                         *Addition takes place only after sterilization pH is adjusted to 7.4 with     NaOH before sterilization  120° C. for 20 minutes                 

LIST OF REFERENCES

Bibb M. et al, Gene 30, 157-166, 1984

Birnboim und Doly, Nucl Acids Res 7, 1513-1523, 1979

Breton A. M. et al, J Bacteriol, 161: 523-528 (1985)

Breton A. M. et al, J Biotechnol, 4: 303-311 (1986)

Breton A. M. und Guespin-Michel J. F., FEMS Microbiol Lett, 40: 183-188(1987)

Datta N. et al, J Bacteriol 108: 1244-1249 (1971)

Haymes B. T. et al, Nucleic Acid Hybridisation a Practical Approach, IRLPress, Oxford, England (1985)

Hedges R. W. and Matthew M., Plasmid 2, 269-278, 1979

Hohn B. und Collins J., Gene 11, 291, 1980

Hopwood et al, "Genetic Manipulation of Streptomyces a LaboratoryManual"; The John Innes Foundation, Norwich UK, 1985

Jaoua S. et al, Plasmid 18: 111-119 (1987 )

Jaoua S. et al, Plasmid 23: 183-193 (1990)

Jaoua S. et al, Plasmid 28: 157-165 (1992)

Kaiser D., Genetics of Myxobacteria, in: "Myxobacteria: Development andCell Interactions", ed E. Rosenberg, pp 163-184, Springer Verlag,Berlin/New York (1984);

Kuner J. M. und Kaiser D., Proc Natl Acad Sci USA, 78: 425-429 (1981)

Kuspa und Kaiser, J Bacteriol Vol 171 (5), 2762-2772 (1989)!

Malpartida und Hopwood Nature 309, 462-464, 1984!.

Maxam and Gilbert, `Sequencing end-labelled DNA with base-specificchemical cleavage`, in: Methods in Enzymology 65: 499-560, AcademicPress, New York, London, (1980).

Maniatis T., Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Cold Spring Habor, N.Y. (1982)

O'Conner K. A. und Zusman D. R., J Bacteriol, 155: 317-329 (1983)

Reichenbach et al, Trends in Bilotechnology, Vol 6 (6), (1988)

Rigby D. W. J. et al, J Mol Biol 113, 237-251, 1977

Sherman D. et al, EMBO J 8, 2717-2725, 1989

Shimkets L. J. et al, Proc Natl Acad Sci USA, 80: 1406-1410 (1983)

Simon R. et al, Bio/Technol. Nov 83, 784-791, 1983

Smith C. L. et al, Methods Enzymol 151, 461-489 (1987)

Schupp T. et al, Gene 64, 179-188, 1988

Schupp T. et al, FEMS Microbiol Letters 36, 237-251, 1986

Wahl G. M. et al, Proc Natl Acad Sci, USA 84, 2160-2164 (1987)

Ward et al, Mol Gen Genet 203, 468-478, 1986

Patent literature

EP-A 0 358 606

U.S. Pat. No. 4,910,140

WO 87/03907

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 784 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Sorangium cellulosum                                            (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Cosmid clone p98/1                                                 (ix) FEATURE:                                                                 (A) NAME/KEY: misc_feature                                                    (B) LOCATION: 1..784                                                          (D) OTHER INFORMATION: /product="Constituent of the                           `soraphen gene cluster`"                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTCGACTACGCCTCCCACTCCGCCCAGATGGACGCCGTCCAAGACGAGCTCGCCGCAGGT60                CTAGCCAACATCGCTCCTCGGACGTGCGAGCTCCCTCTTTATTCGACCGTCACCGGCACC120               AGGCTCGACGGCTCCGAGCTCGACGGCGCGTACTGGTATCGAAACCTCCGGCAAACCGTC180               CTGTTCTCGAGCGCGACCGAGCGGCTCCTCGACGATGGGCATCGCTTCTTCGTCGAGGTC240               AGCCCCCATCCCGTGCTCACGCTCGCCCTCCGCGAGACCTGCGAGCGCTCACCGCTCGAT300               CCCGTCGTCGTCGGCTCCATTCGACGCGACGAAGGCCACCTCGCCCGCCTGCTCCTCTCC360               TGGGCGGAGCTCTCTACCCGAGGCCTCGCGCTCGACTGGAACGCCTTCTTCGCGCCCTTC420               GCTCCCCGCAAGGTCTCCCTCCCCACCTACCCCTTCCAACGCGAGCGCTTCTGGCTCGAC480               GCCTCCACGGCGCACGCTGCCGACGTCGCCTCCGCAGGCCTGACCTCGGCCGACCACCCG540               CTGCTCGGCGCCGCCGTCGCCCTCGCCGACCGCGATGGCTTTGTCTTCACAGGACGGCTC600               TCCCTCGCAGAGCACCCGTGGCTCGAAGACCACGTCGTCTTCGGCATACCCGTCCTGCCA660               GGCGCCGCCCTCCTCGAGCTCGCCCTGCATGTCGCCCATCTCGTCGGCCTCGACACCGTC720               GAAGACGTCACGCTCGACCCCCCCCTCGCTCTCCCATCGCAGGGCGCCGTCCTCCTCCAG780               ATCT784                                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="synthetic oligonucleotide                            Oli1"                                                                         (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TACTGGTATCGAAACCT17                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="synthetic oligonucleotide                            Oli2"                                                                         (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAAGACGACGTGGTCTT17                                                           __________________________________________________________________________

We claim:
 1. An isolated DNA molecule that encodes a polypeptiderequired for the biosynthesis of soraphen A, wherein said DNA moleculespecifically hybridizes to SEQ ID NO:
 1. 2. The isolated DNA moleculeaccording to claim 1, wherein said DNA molecule is obtained from thegenome of a myxobacterium.
 3. The isolated DNA molecule according toclaim 2, wherein said DNA molecule is obtained from the genome ofSorangium cellulosum.
 4. The isolated DNA molecule according to claim 1,wherein said DNA molecule comprises SEQ ID NO:
 1. 5. An isolated DNAmolecule consisting of SEQ ID NO:
 1. 6. A prokaryotic vector comprisingthe isolated DNA molecule according to claim
 1. 7. The vector accordingto claim 6, which is a transformation vector.
 8. The vector according toclaim 6, which is an expression vector.
 9. A prokaryotic host cellcomprising the vector according to claim
 6. 10. A cosmid clonedesignated p98/1 and deposited as NRRL B-21255.